In Vivo and Ex Vivo Expansion of Hematopoietic Stem Cells With a Targeted Combination of Clinically Tested, FDA Approved Drugs

ABSTRACT

The present invention provides a therapeutic approach to maintain and expand HSCs in vivo using currently available medications that target GSK-3 and mTOR. The present invention also provides a system and method for the ex vivo culturing of HSCs, where an mTOR inhibitor is combined with a GSK-3 inhibitor within the culturing conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/274,601, filed Sep. 23, 2016, which is a continuation ofU.S. patent application Ser. No. 14/673,125, filed Mar. 30, 2015,granted, which is a divisional of U.S. patent application Ser. No.13/084,180, filed Apr. 11, 2011, granted, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 61/323,203, filedApr. 12, 2010, the contents of which are each incorporated by referenceherein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R01MH58324 awardedby the National Institute of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

It is well accepted that stem cells possess the unique ability to selfrenew and differentiate into a diverse range of specialized cell types.For example, hematopoietic stem cells (HSCs) have provided an importantwindow into stem cell biology as well as a valuable clinical tool fortreatment of hematopoietic malignancies and other disorders. However,the complex signaling network regulating the balance between HSC selfrenewal and differentiation is still not well understood.

One important regulator of HSC homeostasis is suggested by the highlyprevalent clinical finding that therapeutic lithium increasescirculating HSCs (as CD34⁺ cells; Ballin, et al. 1998. Br. J. Haematol.100:219-221) and peripheral blood counts (Boggs, et al., 1983, Semin.Hematol. 20:129-138; Joyce, 1984, Br. J. Haematol. 56:307-321; Ricci, etal. 1981, Haematologica. 66:627-633) in greater than 90% of patientstaking lithium, and the laboratory findings that lithium also increasestransplantable HSCs in mice (Boggs, et al., 1983. Semin. Hematol.20:129-138; Joyce, 1984, Br. J. Haematol. 56:307-321). Because lithiumdirectly inhibits glycogen synthase kinase-3 (GSK-3) (GSK-3; Klein, etal., 1996, Proc. Natl. Acad. Sci. U.S.A 93:8455-8459), activatingcritical signaling pathways such as the Wnt and PI3K/PTEN/Akt pathways(Hedgepeth, et al., 1997, Dev. Biol. 185:82-91; Stambolic, et al., 1996,Curr. Biol. 6:1664-1668), these clinical and laboratory observationsimplicate GSK-3 as an important regulator of HSC homeostasis (Hedgepeth,et al., 1997, Dev. Biol. 185:82-91; Phiel, et al., 2001, Annu. Rev.Pharmacol. Toxicol. 41:789-813; Focosi, et al., 2009, J. Leukoc. Biol.85:20-28). Support for this hypothesis comes from pharmacologicalstudies showing that HSCs and hematopoietic progenitor cells (HPCs) areincreased, and hematopoietic repopulation is enhanced when BM transplantrecipient mice are treated with alternative GSK-3 inhibitors(Trowbridge, et al., 2006. Nat. Med. 12:89-98; Goessling, et al., 2009,Cell. 136:1136-1147; Holmes, et al., 2008, Stem Cells. 26:1288-1297).Furthermore, mouse ES cells treated with GSK-3 inhibitors maintainpluripotency (Sato, et al., 2004, Nat. Med. 10:55-63; Ying, et al.,2008, Nature. 453:519-523), and mouse ES cells lacking Gsk3a and Gsk3bmaintain expression of markers of pluripotency under conditions thatinduce control ES cells to differentiate (Doble, et al., 2007, Dev.Cell. 12:957-971). These observations suggest a negative role for GSK-3in ESC renewal. However, Gsk-3 loss of function in HSCs has notpreviously been performed, and the downstream pathways regulated byGSK-3 in HSCs have not yet been established.

Canonical Wnt signaling, which inhibits GSK-3 and thereby stabilizesbeta-catenin, plays a central role in the self renewal of diverse stemcell populations (Sato, et al., 2004, Nat. Med. 10:55-63; Doble, et al.,2007, Dev. Cell. 12:957-971; Staal, et al., 1999, Int. Immunol.11:317-323; Reya, et al., 2005, Nature. 434:843-850; Staal, et al.,2008, Eur. J. Immunol. 38:1788-1794; Malhotra, et al., 2009, Cell StemCell. 4:27-36). A role for Wnt signaling in hematopoiesis is supportedby observations that Wnt ligands enhance proliferation of HSCs ex vivo(Austin, et al., 1997, Blood. 89:3624-3635; Van Den Berg, et al., 1998,Blood. 92:3189-3202; Willert, et al., 2003, Nature. 423:448-452) andthat Wnt antagonists inhibit HSC proliferation and reconstitution (Reya,et al., 2003, Nature. 423:409-414; Jeannet, et al., 2008, Blood.111:142-149). In addition, overexpression of stabilized β-cateninpromotes HSC self renewal and proliferation ex vivo under certainconditions (Austin, et al., 1997, Blood. 89:3624-3635; Van Den Berg, etal., 1998, Blood. 92:3189-3202; Willert, et al., 2003, Nature.423:448-452; Reya, et al., 2003, Nature. 423:409-414; Baba, et al.,2006, J. Immunol. 177:2294-2303), and conditional deletion of β-cateninusing vav-cre impairs HSC function in competitive repopulation assays(Zhao, et al., 2007, Cancer Cell. 12:528-541). Furthermore, long-termreconstituting capacity in serial transplants is impaired in HSCsrecovered from fetal liver of Wnt3a KO embryos (Austin, et al., 1997,Blood. 89:3624-3635; Van Den Berg, et al., 1998, Blood. 92:3189-3202;Willert, et al., 2003, Nature. 423:448-452; Reya, et al., 2003, Nature.423:409-414; Baba, et al., 2006, J. Immunol. 177:2294-2303; Luis, etal., 2009, Blood. 113:546-554) or from adults overexpressing the Wntinhibitor Dkk in the hematopoietic niche (Fleming, et al., 2008, CellStem Cell. 2:274-283), which suggests that Wnt signaling is required tomaintain the long-term repopulating activity of HSCs.

However, there are conflicting reports on the requirement forWnt/β-catenin signaling in basal hematopoiesis: conditional disruptionof β-catenin and γ-catenin/plakoglobin in adult HSCs does not affecttheir ability to self renew and reconstitute hematopoietic lineages(Jeannet, et al., 2008, Blood. 111:142-149; Cobas, et al., 2004, J. Exp.Med. 199:221-229; Koch, et al., 2008, Blood. 111:160-164). In addition,although overexpression of stabilized β-catenin increasesimmunophenotypic HSCs, this is associated with a loss of repopulatingactivity and hematopoietic failure in vivo (Scheller, et al., 2006, Nat.Immunol. 7:1037-1047; Kirstetter et al., 2006, Nat. Immunol.7:1048-1056), findings that appear incompatible with a positive role forβ-catenin in hematopoiesis. A general conclusion from these apparentlyconflicting reports is that the role of Wnt signaling in hematopoiesisis complex and context dependent (Staal, et al., 2008, Eur. J. Immunol.38:1788-1794; Malhotra, et al., 2009, Cell Stem Cell. 4:27-36). However,although the β-catenin loss-of-function studies suggest that canonicalWnt signaling is not essential for basal hematopoiesis in adults, theydo not rule out a possible role for the Wnt/β-catenin pathway undernonbasal conditions, and are still compatible with gain-of-functionexperiments in which the pathway is activated.

GSK-3 is also inhibited by Akt/PKB, which in turn requires the activityof PI3K and is antagonized by phosphatase and tensin homolog (PTEN), aPI3 phosphatase. Loss of Pten transiently increases HSCs, which isfollowed by progressive HSC depletion, increased lineage commitmentresembling myeloproliferative disorder, and acute leukemia (Yilmaz, etal., 2006, Nature. 441:475-482; Zhang, et al., 2006, Nature.441:518-522). This expansion and subsequent depletion in Pten KO HSCs ismediated through mammalian target of rapamycin (mTOR), as the phenotypeis reversed by treatment with rapamycin (Yilmaz, et al. 2006, Nature.441:475-482), and a similar HSC phenotype is observed with KO oftuberous sclerosis complex 1 (Tsc1), a negative regulator of mTOR (Chen,et al., 2008, J. Exp. Med. 205:2397-2408; Gan, et al., 2008, Proc. Natl.Acad. Sci. U.S.A 105:19384-19389). As GSK-3 is an indirect target ofPTEN and antagonizes mTOR through phosphorylation of Tsc2 (Inoki, etal., 2006, Cell. 126:955-968), inhibition of GSK-3 could mimic thehematopoietic phenotype of Pten and Tsc1 KOs.

Currently available pharmacological data from humans and mice suggestthat GSK-3 is an important regulator of HSC homeostasis, but thepathways regulated by GSK-3 in HSCs have not been defined. Furthermore,Gsk-3 loss of function in HSCs/HPCs has not previously been reported foreither Gsk3a or Gsk3b, and this is an essential step to defining therole of Gsk-3 within HSCs.

Without question, the maintenance and expansion of long-termtransplantable HSCs under in vivo and ex vivo conditions is a crucialcomponent and major challenge in stem cell research and therapeutichematopoietic stem cell transplantation. Thus, the ability to maintainand expand HSCs in culture has long been deemed the holy grail ofhematopoietic stem cell research. Unfortunately, the ability to expandHSCs consistently and effectively in ex vivo culture conditions has notyet been perfected. Therefore, there is a long felt need in the art fora culturing system and method for maintaining and expanding HSCs. Byunderstanding the complex signaling network regulating the balancebetween HSC self renewal and differentiation, the present inventionsatisfies this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a method for the maintenance and expansion of ahematopoietic stem cell (HSC). The method includes culturing the HSC ina medium including at least one glycogen synthase kinase-3 (GSK-3)inhibitor and at least one mammalian target of rapamycin (mTOR)inhibitor, wherein the multipotentiality of the HSC is retained duringthe culturing.

In one embodiment, the HSC is derived from a mammal. In anotherembodiment, the mammal is a human. In yet another embodiment, exogenousgenetic material is introduced into the HSC. In yet another embodiment,the medium further includes at least one cytokine. In yet anotherembodiment, the medium further includes a promotion factor. In yetanother embodiment, the GSK-3 inhibitor is lithium, or a salt thereof.In yet another embodiment, the mTOR inhibitor is rapamycin. In yetanother embodiment, the culturing is ex vivo.

The invention also includes an isolated HSC prepared by a method ofculturing the HSC in a medium comprising at least one GSK-3 inhibitorand at least one mTOR inhibitor, wherein the multipotentiality of theisolated HSC is retained during the culturing.

The invention further includes a method of treating a mammal having adisease, disorder or condition. The method includes obtaining anisolated HSC from a donor, culturing the HSC in a medium comprising atleast one GSK-3 inhibitor and at least one mTOR inhibitor, andadministering the cultured HSC to the mammal.

In one embodiment, the mammal is a human. In another embodiment, theisolated HSC is allogeneic with respect to the mammal. In yet anotherembodiment, the isolated HSC is autologous with respect to the mammal.In yet another embodiment, the disease, disorder or condition isselected from the group consisting of a genetic disease, acutelymphoblastic leukemia, acute myeloblastic leukemia, chronic myelogenousleukemia (CIVIL), Hodgkin's disease, multiple myeloma, non-Hodgkin'slymphoma, anemia, aplastic anemia, beta-thalassemia, Blackfan-Diamondsyndrome, globoid cell leukodystrophy, sickle-cell anemia, severecombined immunodeficiency, X-linked lymphoproliferative syndrome,Wiskott-Aldrich syndrome, Hunter's syndrome, Hurler's syndrome, LeschNyhan syndrome, and osteopetrosis. In yet another embodiment, thecultured HSC administered to the mammal remains present and/orreplicates in the mammal. In yet another embodiment, prior toadministering the HSC, the HSC is cultured ex vivo in the medium. In yetanother embodiment, prior to administering the HSC, the HSC isgenetically modified.

The invention further includes a culturing medium for expanding andmaintaining HSC. The culturing medium includes at least one GSK-3inhibitor and at least one mTOR inhibitor, wherein the multipotentialityof the HSC is retained while the HSC is maintained or expanded in theculturing medium.

The invention further includes a method for the in vivo maintenance andexpansion of HSC in a mammal. The method includes transplanting the HSCinto a tissue of the mammal and administering to the mammal at least oneGSK-3 inhibitor and at least one mTOR inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1, comprising FIG. 1A-1E, demonstrates that lithium and other GSK-3inhibitors expand HSC/HPCs. FIG. 1A depicts the absolute number ofLineage-Sca-1+cKit+(LSK) fraction, which is enriched for HSCs, andimmunophenotypic LT-HSC (LSK; CD34-Flk2-) and ST-HSC (LSK CD34+Flk-2-)fractions in bone marrow harvested from mice treated with control orlithium diet for 2 weeks. FIG. 1B depicts the cellularity of bonemarrow, thymus and spleen in control and lithium treated mice, shown asthe number of nucleated cells/mouse recovered from both femurs andtibias, thymus, and spleen. FIG. 1C depicts the percentage (left) andabsolute number (right) of HSC/HPCs (as LSK) in mice treated with 6BIOvs control for two weeks. FIG. 1D depicts a colony formation assay:Purified total c-Kit+ cells were treated with lithium, 6BIO, AR-A014418,or Ru (1-OH) (also known as DW12) for 3 days and plated inmethylcellulose with hematopoietic cytokines for 12 days. Coloniesof >30 viable cells were counted and the mean colony number/50,000plated cells for each of three separate experiments is shown. FIG. 1Edepicts the total numbers of c-Kit+ cells in each drug treatment after 3days. The primary cultures are shown.

FIG. 2, comprising FIGS. 2A-2H, demonstrates that Gsk-3 depletionexpands HSCs and HPCs in primary bone marrow (BM) transplants. Asdepicted in FIG. 2A, irradiated mice were reconstituted with BM aftertransduction with lentivirus with or without Gsk3-rnai. Peripheral bloodwas examined 20 weeks after transplantation, where numbers withinhistograms indicate percent GFP⁺ cells. One representative of fivesimilar experiments is illustrated, and similar results were obtainedwith Gsk3-rnai-C4. FIG. 2B depicts GFP⁺ myeloid cells (Gr1⁺CD11b⁺) inperipheral blood for 10 control and 9 Gsk3-rnai-C2 recipients after BMtransplantation (BMT; arrow). Depicted in FIG. 2C are immunoblots forGSK-3alpha/beta and beta-catenin in BM from primary recipients 16 weeksafter transplantation. Data represent independent replicates from 6control and 6 Gsk3-rnai recipients. FIG. 2D depicts the percent GFP⁺ LSKcells in control and Gsk3-rnai-C2 primary recipients. FIG. 2E depictsabsolute number of GFP⁺ LSK, LSK CD34⁻Flk2⁻, and LSK CD34⁺Flk2⁻ cells.FIG. 2F depicts representative FCM showing GFP⁺ cells in theHSC-containing LSK fraction (red gate) for control and Gsk3-rnai-C2primary recipients. FIG. 2G depicts representative FCM using SLAMmarkers, where the difference between control and Gsk3-rnai wassignificant (P<0.05). The numbers in FIGS. 2F and 2G indicate percentcells within gates. FIG. 2H depicts colony formation using GFP⁺ cellsplated in methylcellulose with cytokines and scored for CFU-C. Datarepresent mean colonies per well performed in duplicate groups for 5mice per construct repeated in 3 separate experiments. *P<0.05 versusrespective control value.

FIG. 3, comprising FIGS. 3A-D, depicts annexin V staining andcellularity of bone marrow harvested from transplant recipients. FIG. 3Adepicts a flow cytometric detection of Annexin V using bone marrow cellsharvested from 1° recipients after 4 month bone marrow transplantationfrom both control and Gsk3 RNAi. Annexin V was measured in the GFP+LSKgated population. FIG. 3B depicts the total number of bone marrow cellsand number of GFP+ cells recovered in bone marrow from 1° recipientsafter 4 month transplant. FIG. 3C depicts the total number and number ofGFP+ cells recovered in bone marrow from 2° recipients after 4 months.FIG. 3D depicts the total number and number of GFP+ cells recovered inbone marrow from 3° recipients after 4 months.

FIG. 4, comprising FIGS. 4A and 4B, demonstrates that Gsk-3 knockdownincreases cycling of the HSC-enriched LSK cell population. To assesscell cycle status of the HSC-enriched LSK population, sorted GFP⁺ LSKand GFP⁺ LSK Flk2⁻ cells from primary recipients of control andGsk3-rnai 4 months after BM transplantation were stained with Hoechstand Pyronin and analyzed by FCM, as depicted in FIG. 4A. RepresentativeFACS data are shown for control versus Gsk3-rnai-C2. As depicted in FIG.4B, at 4 months after BM transplantation, primary recipients of controland Gsk3-rnai were fed BrdU in the drinking water for 7 days. SortedGFP⁺ LSK Flk2⁺ and Flk2⁻ cells were stained with BrdU-APC antibody andPI to analyze BrdU incorporation. Representative FACS data are shown forcontrol versus Gsk3-rnai-C2. Similar results were obtained by BrdUversus 7-AAD staining (BD). Percent cells are shown for the indicatedgates and quadrants. In FIGS. 4A and 4B, lower left gate represents Go,upper left represents G₁, and upper right represents S, G₂, and M phasesof the cell cycle, as shown in the diagram in FIG. 4A.

FIG. 5, comprising FIGS. 5A-C, demonstrates that Gsk3-depleted HSCs arefunctionally deficient. As depicted in FIG. 5A, limiting dilutionexperiments were performed with 4 doses (x axis) of GFP⁺ test BM fromvector-control and Gsk3-rnai primary recipients (4 donors per group)combined with a fixed number (2×10⁵) unlabeled competing cellstransplanted into groups of at least 5 recipients per dose. Chimerism at4 months after transplantation for each dose is represented as thepercentage of GFP⁺ cells in BM for control and Gsk3-rnai. Depicted inFIG. 5B are percent donor-derived immunophenotypic HSCs/HPCs (as GFP⁺LSK cells) in the 1×10⁶ test cell group 4 months after transplantation.Depicted in FIG. 5C are absolute number of donor-derivedimmunophenotypic HSCs/HPCs (as GFP⁺ LSK cells) in the 1×10⁶ test cellgroup 4 months after transplantation.

FIG. 6, comprising FIGS. 6A-6H, demonstrates that Gsk-3 knockdowndepletes HSCs in serial BM transplants. As depicted in FIG. 6A,noncompetitive serial transplants were performed by transplanting 2×10⁵sorted GFP⁺ cells from primary recipients of control or Gsk3-rnaitransduced BM into lethally irradiated recipients (10 mice per group).Survival of secondary recipients receiving control or Gsk3-rnai BM isshown as a Kaplan-Meier plot. FIG. 6B depicts percent HSC-containing LSKfraction in control and Gsk3-rnai secondary recipients. FIG. 6C depictsabsolute number of GFP⁺ LSK, LSK CD34⁻Flk2⁻, and LSK CD34⁺Flk2⁻ cells incontrol and Gsk3-rnai secondary recipients. FIG. 6D depictsrepresentative FCM data, presented as the distribution of CD34⁻Flk2⁻,CD34⁺Flk2⁻, and CD34⁺Flk2⁻, which immunophenotypically correspond toLT-HSCs, ST-HSCs, and MPPs in the LSK population, from control andGsk3-rnai secondary recipients. Percent cells are shown for theindicated gates. As depicted in FIG. 6E, a colony formation assay withsorted GFP⁺ cells from control and Gsk3-rnai secondary recipient BM wasperformed and scored as in FIG. 2 using GFP⁺BM from 5 control and 5Gsk3-rnai mice. As depicted in FIG. 6F, the frequencies of commonmyeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), andmegakaryocyte-erythroid progenitor (MEP) cells were measured bydetection of CD16/32 and CD34 expression in the lineage⁻sca-1⁻c-kit⁺gated population. The common lymphoid progenitor (CLP) fraction wasmeasured as CD127⁺ cells in the lineage⁻sca-1^(lo)c-kit^(lo) gate. FIG.6G depicts lethally irradiated mice were reconstituted with 4×10⁵ sortedGFP⁺BM cells from secondary recipients of vector or Gsk3-rnai transducedBM. The Kaplan-Meier survival curve shows the survival of tertiaryrecipients of BM from control or Gsk3-rnai mice. FIG. 6H depictsabsolute number of immunophenotypic HSCs/HPCs, as LSK cells, in controland Gsk3-rnai tertiary recipients. *P<0.05.

FIG. 7, comprising FIGS. 7A-7C, demonstrates that β-catenin is requiredfor the increase in HSCs/HPCs induced by Gsk3-rnai. As depicted in FIG.7A, BM cells were harvested from Mx-Cre;β-catenin^(fl/fl) mice with orwithout injection of polyI:polyC for 14 days, transduced with control orGsk3-rnai carrying lentivirus, and transplanted into lethally irradiatedrecipient mice. After 4 months, percentage and absolute number ofHSC-containing LSK fraction were compared among the 4 groups. Asdepicted in FIG. 7B, BM cells were harvested at 4 months from primaryrecipients of WT and Mx-Cre;β-catenin^(fl/fl) mice transduced withvector control or Gsk3-rnai lentivirus (from primary recipient mice inFIG. 7A) and transplanted into lethally irradiated secondary hosts.After 4 months, percentage and absolute number of HSC-containing LSKfraction were compared among the 4 groups. FIG. 7C depicts a summary ofserial transplantation data in WT versus β-catenin CKO mice. Shown isfold change in GFP⁺ LSK cells in recipients of Gsk3-depleted BMnormalized to vector control, for otherwise WT primary, secondary, andtertiary recipients as well as for primary and secondary β-catenin CKOrecipients. Survival in tertiary recipients of Gsk3/β-catenin-deficientBM was too low for statistical significance. *P<0.05.

FIG. 8, comprising FIGS. 8A and 8B, depicts increased colony formationinduced by Gsk3-rnai requires B-catenin. A colony formation assay wasperformed using sorted GFP+ cells harvested from 1° recipients (FIG. 8A)or 2° recipients (FIG. 8B) of wild-type (left) and β-catenin KO (right)marrow transduced with control (open boxes) or Gsk3-rnai (filled boxes)lentivirus. Data represent mean number of colonies per well for fivemice per construct repeated in three separate experiments.

FIG. 9, comprising FIGS. 9A-9H, demonstrates increased HSCs/HPCs inrapamycin-treated recipients of Gsk3-depleted BM and increased survivalof tertiary recipients receiving Gsk3-depleted BM from rapamycin treated2° hosts. As depicted in FIG. 9A, BM was harvested fromMx-Cre;β-catenin^(fl/fl) mice treated with or without polyI:polyC,transduced with control or Gsk3-rnai lentivirus, and transplanted intoirradiated recipients. After 4 months, GFP⁺ cells were sorted (pooledfrom 5 mice per group), and phospho-ribosomal protein S6 (p-S6) wasdetected in cell lysates by immunoblot. FIG. 9B depicts flow cytometricdetection of phospho-ribosomal protein S6 with BM cells in FIG. 9A. Asdepicted in FIG. 9C, irradiated mice were reconstituted with BMtransduced with control vector or Pten-rnai. After 4 months,phospho-GSK-3 and phospho-ribosomal protein S6 were assessed in GFP⁺cells by immunoblot. As depicted in FIG. 9D, NIH3T3 cells were infectedfor 3 days with control or Pten-rnai lentivirus, and phospho-GSK-3α/β incontrol and Pten-depleted cells was detected by immunoblot. FIG. 9Edepicts increased percentage of LSK cells in bone marrow of 2°recipients of Gsk3-depleted BM that were treated with rapamycin for 8weeks. GFP⁺ cells (2×10⁶) from primary recipients of control orGsk3-rnai were transplanted into 10 irradiated recipients per group.After 1.5-2 months, secondary recipients were injected with rapamycin orvehicle every other day for 8 weeks. Percent GFP⁺ LSK cells was comparedamong the 4 groups. FIG. 9F depicts absolute number of GFP⁺ LSK cells asin FIG. 9E. FIG. 9G depicts colony formation with sorted GFP⁺ cells fromFIG. 9E. FIG. 9H depicts a Kaplan-Meier plot showing survival oftertiary recipients transplanted with BM from control- andrapamycin-treated secondary recipients in FIGS. 9E-9G. Illustrated iscontrol vector BM from secondary recipients treated with vehicle orrapamycin and Gsk3-rnai-infected BM from secondary recipients treatedwith vehicle or rapamycin transplanted to lethally irradiated tertiaryrecipients. *P<0.05.

FIG. 10, comprising FIGS. 10A-10D, demonstrates that Gsk3b KO depletesHSCs in serial BM transplants. Noncompetitive serial transplants wereperformed by transplanting 4×10⁵ fetal liver cells (CD45.2) from E17.5WT, Gsk3b^(+/−), and Gsk3b^(−/−) embryos into lethally irradiatedrecipient mice (CD45.1). FIGS. 10A and 10B depict reconstitution ofperipheral blood, including B cells (B220⁺), T cells (CD3⁺), and myeloidcells (Mac-1⁺G-R-1⁺, in primary (FIG. 10A) and secondary (FIG. 10B)recipients of WT, Gsk3b^(+/−), and Gsk3b^(−/−) fetal liver cells.Secondary transplants were performed after 16 weeks of engraftment bypooling BM from 3-4 reconstituted primary recipients to transplant 4×10⁵whole BM cells into lethally irradiated CD45.1 secondary recipients (10hosts per genotype). As depicted in FIGS. 10C and 10D, percentage of LSKcells in CD45.2⁺BM was compared among recipients of WT, Gsk3b^(+/−), andGsk3b^(−/−) fetal liver cells in primary (FIG. 10C) and secondary (FIG.10D) hosts. *P<0.05.

FIG. 11 demonstrates that GSK-3 functions in 2 major pathways toregulate HSC self renewal and lineage commitment. Inhibition of GSK-3activates Wnt and mTOR signaling. In the canonical Wnt pathway, GSK-3and β-catenin bind to the Axin complex, along with APC. GSK-3phosphorylates β-catenin, targeting it for rapid destruction. Wntbinding to the Fz/Lrp receptor complex causes inhibition of GSK-3, whichin turn stabilizes β-catenin and activates Wnt target genes that promoteprogenitor proliferation and self renewal. In PI3K/PTEN-regulatedpathways, growth factors (GFs) bind to surface receptors and activatePI3K, leading to activation of Akt, whereas PTEN inhibits activation ofAkt. Once activated, Akt phosphorylates and inhibits GSK-3. GSK-3phosphorylates Tsc2, inhibiting the mTOR pathway. Thus, inhibition ofGSK-3 activates mTOR and promotes proliferation and exit from the LT-HSCpool. Inhibition of GSK-3 thus activates distinct downstream signalingpathways that have opposing functions in HSC renewal anddifferentiation.

FIG. 12, comprising FIGS. 12A and 12B, demonstrates the effect ofB-catenin conditional knockout on colony formation in Gsk3-depleted bonemarrow. A colony formation assay was performed using sorted GFP+ cellsfrom each group in 1° (FIG. 12A) and 2° (FIG. 12B) recipients. Datarepresent mean number of colonies per well for five mice per constructrepeated in three separate experiments.

FIG. 13 demonstrates ex vivo mouse HSC culturing and transplantation.c-kit+ cells were isolated from adult mouse bone marrow and cultured inserumfree, cytokine-free defined medium for 7 days with (triangle) orwithout (circle) our formulation. After 7 days, the entire culture wastransplanted to lethally irradiated mice (5/group) and survival wasmonitored over more than 16 weeks. All control animals (cultured withoutadditives or no transplant) died within 17 days. Mice receiving bonemarrow cells cultured in cytokine-free, serum-free medium with additivessurvived over 16 weeks. The experiment has been repeated 3 times.Erythrocytic, myelocytic, lymphocytic, and megakaryocytic lineages werepresent in peripheral blood and bone marrow after 4 months.

FIG. 14 demonstrates ex vivo culturing of HSCs from human umbilical cordblood. CD34+ cells from human UCB were isolated and either transplantedimmediately to NSG mice (sample 1) or cultured in the cytokine-free,serum-free medium alone (sample 2), supplemented with our non-proteinadditives (sample 3), or with conventional cytokine cocktail (sample 4,includes IL3, SCF, FL, and Tpo) for either 4 days (shown) or 7 days (notshown). CD34+ cells increased 2-3 fold after one week when cultured withadditives compared to no additives. Samples 2-4 were transplanted to NSGmice. Mice were bled monthly for flow cytometric analysis of human/mousechimerism in peripheral blood (not shown) and were sacrificed at 4months for flow cytometric analysis of human/mouse chimerism in bonemarrow. The percent of human CD45.1 over total mononuclear cells in thebone marrow effluent is shown for each group. There were 5 mice pergroup and the experiment has been repeated with similar results 3 times.Secondary transplantation showed that human cells in group 3, but noneof the other groups, sustained long-term repopulating activity formyelocytic, erythrocytic, and lymphocytic lineages.

DETAILED DESCRIPTION

The present invention provides a therapeutic approach to expand HSCs invivo using currently available medications that target GSK-3 and mTOR.The present invention also provides a system and method for the ex vivoculturing of HSCs, where an mTOR inhibitor is combined with a GSK-3inhibitor within the culturing conditions. The present invention isbased on the discovery that Gsk-3 loss of function coupled withinhibition of mTOR provides for improved maintenance and expansion ofHSCs in vivo and ex vivo. It is demonstrated herein that knockdown ofGsk-3a/b (hereafter, Gsk-3 is used to refer to both genes) initiallyexpand the HSC-enriched pool of lineage⁻sca-1⁺c-kit⁺ (LSK) cells,similar to the effects of lithium and other GSK-3 inhibitors, and thisrequires endogenous β-catenin function. However, in assays of long-termstem cell function, Gsk3-deficient HSCs are progressively depleted,revealing an unexpected positive role for GSK-3 in the maintenance ofHSC self renewal. Furthermore, the data suggest that GSK-3 functionsdownstream of PTEN to antagonize mTOR signaling in phenotypic HSCs(HSC-enriched LSK population) in addition to its role in antagonizingWnt/β-catenin signaling. Based on these observations, Gsk-3 loss offunction coupled with inhibition of mTOR expands phenotypic HSCs invivo. These findings point to a critical role for GSK-3 in regulatingthe decision between self renewal and differentiation in HSCs.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

“Allogeneic” refers to a graft derived from a different animal of thesame species. As used herein, the term “autologous” is meant to refer toany material derived from the same individual to which it isre-introduced.

The terms “precursor cell,” “progenitor cell,” and “stem cell” are usedinterchangeably in the art and herein and refer either to a pluripotent,or lineage-uncommitted, progenitor cell, which is potentially capable ofan unlimited number of mitotic divisions to either renew itself or toproduce progeny cells which will differentiate into the desired celltype. Unlike pluripotent stem cells, lineage-committed progenitor cellsare generally considered to be incapable of giving rise to numerous celltypes that phenotypically differ from each other. Instead, progenitorcells give rise to one or possibly two lineage-committed cell types.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. Thus, a substantially purifiedcell refers to a cell which has been purified from other cell types withwhich it is normally associated in its naturally occurring state.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to a tissue, a stem cell, a neural stem cell, a skincell, bone marrow, and solid organs such as heart, pancreas, kidney,lung and liver.

As used herein, a “therapeutically effective amount” is the amount of atherapeutic composition, such as a GSK-3 and/or mTOR inhibitor,sufficient to provide a beneficial effect to a mammal or culture orculturing system to which the composition is administered.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced from or produced outsidean organism, cell, or system.

“Expandability” is used herein to refer to the capacity of a cell toproliferate for example to expand in number, or in the case of a cellpopulation, to undergo population doublings.

“Graft” refers to a cell, tissue, organ or otherwise any biologicalcompatible lattice for transplantation.

As used herein, the term “growth medium” is meant to refer to a culturemedium that promotes growth of cells.

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e. increasing, decreasing, and the like.

“Proliferation” is used herein to refer to the reproduction ormultiplication of similar forms, especially of cells. That is,proliferation encompasses production of a greater number of cells, andcan be measured by, among other things, simply counting the numbers ofcells, measuring incorporation of ³H-thymidine into the cells, and thelike.

As used herein, a “promotion factor” refers to any molecule that assistsin maintaining and/or proliferating cells.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains componentssuch as, without limitation, a nucleic acid, peptide, cells, one or moreinhibitors and/or composition of the invention or be shipped togetherwith a container which contains such components. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the instructional material and the components be usedcooperatively by the recipient.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

Hematopoietic stem cell (HSC) homeostasis depends on the balance betweenself renewal and lineage commitment, but what regulates this decision isnot well understood. Using loss-of-function approaches in mice, it isdemonstrated herein that glycogen synthase kinase-3 (Gsk-3) plays apivotal role in controlling the decision between self renewal anddifferentiation of HSCs. Disruption of Gsk-3 in bone marrow (BM)transiently expanded phenotypic HSCs in a β-catenin-dependent manner,consistent with a role for Wnt signaling in HSC homeostasis. However, inassays of long-term HSC function, disruption of Gsk-3 progressivelydepleted HSCs through activation of the mammalian target of rapamycin(mTOR). This long-term HSC depletion was prevented by mTOR inhibition,and exacerbated by β-catenin knockout. Thus, the present invention isbased on the discovery that GSK-3 regulates both Wnt and mTOR signalingin mouse HSCs, where these pathways promote HSC self renewal and lineagecommitment, respectively, such that inhibition of Gsk-3 in the presenceof rapamycin expands the HSC pool in vivo. Therefore, the presentinvention provides a therapeutic approach to expand HSCs in vivo usingcurrently available medications that target GSK-3 and mTOR, and providesa compelling explanation for the clinically prevalent hematopoieticeffects observed in individuals prescribed the GSK-3 inhibitor lithium.

The present invention also provides a system and method for the ex vivoculturing of HSCs, where an mTOR inhibitor is combined with a GSK-3inhibitor within the culturing conditions. By simultaneously inhibitingGSK-3 and mTOR, HSCs can survive and increase in number within ex vivoculturing conditions. As contemplated herein, the present invention mayalso allow for expansion of human umbilical cord blood HSCs or adultdonor HSCs, and improve the frequency of engraftment and shortened thetime to hematopoietic recovery in recipient bone marrow transplantpatients. The advantage of cord blood is that this can be transplantedwith markedly reduced risk of graft versus host disease and thepotential for reduced immunosuppressive therapy of bone marrowtransplant recipients. The present invention also bypasses the need forexpensive cytokines to diminish the potential side effects associatedwith introducing foreign DNA into bone marrow cells. As contemplatedherein, treatment may include ex vivo culture or included in thetreatment regiment for transplant recipients.

Source of HSC

The classic source of HSCs is bone marrow (BM). In an embodiment of thepresent invention, HSCs may be harvested from BM. BM cells may becollected by puncturing a bone and flushing out the bone marrow cellswith a syringe. About 1 in every 100,000 cells in the marrow is along-term, blood-forming stem cell. Other cells present include stromalcells, stromal stem cells, blood progenitor cells, and mature andmaturing white and red blood cells.

In another embodiment, donor cells may be harvested from peripheral,circulating blood. While a small number of stem and progenitor cellscirculate in the bloodstream, improved cell counts may be obtained byinjecting the donor with a cytokine, such as granulocyte-colonystimulating factor (GCSF). The donor is injected with GCSF a few daysbefore the cell harvest. To collect the cells, an intravenous tube isinserted into the donor's vein and the blood is passed through afiltering system that extracts out CD34⁺ white blood cells and returnsthe red blood cells to the donor. Of the CD34⁺ cells collected,approximately 5 to 20 percent may be HSCs, where the remaining cells area mixture of stem cells, progenitors, and white blood cells of varyingdegrees of maturity.

In other embodiments, blood from the human umbilical cord and placentamay be collected and used as a rich source of HSCs. In still otherembodiments, developing blood-producing tissues of fetal animals mayprovide a source of HSCs for research purposes, as hematopoietic cellsappear early in the development of all vertebrates. As contemplatedherein, the there is no limitation to the source of HSCs, the manner inwhich they are harvested, or the timing of such harvesting.

HSC Culturing Conditions

According to an aspect of the present invention, an ex vivo culturesystem and process for the maintenance and expansion HSCs is provided,such that the expanded cells can be engrafted into patients withoutlosing their capability for multilineage differentiation andreconstitution. HSCs have the capability of both self-renewal and theability to differentiate into distinct hematopoietic cell lineages, suchas myeloid, B-cell and T-cell lineages. As contemplated herein, the exvivo maintenance and expansion of HSCs can be achieved by culturing HSCsin the presence of GSK-3 and mTOR inhibitors.

In one embodiment of the present invention, mammalian HSCs, preferablyhuman HSCs, can be expanded ex vivo by culturing isolated HSCs in aculture medium which comprises the combination of at least one GSK-3inhibitor and at least one mTOR inhibitor. In addition to the at leastone GSK-3 inhibitor and at least one mTOR inhibitor, the culture mediumfurther comprises any culture medium suitable for culturing HSCs. Insome embodiments, the culturing medium may include serum, while in otherembodiments, the culturing medium may be serum-free. Standard culturemedia for HSCs typically contains a variety of essential componentsrequired for cell viability, including inorganic salts, carbohydrates,hormones, essential amino acids, vitamins, and the like. Preferably, theconditions for culturing the HSCs should be as close to physiologicalconditions as possible. The culturing medium, as contemplated herein,may include components that are known to those of ordinary skill in theart and may comprise such components as RPMI 1640, HEPES, FCS, andcommon antibiotics. Any commercially available HSC culturing medium maybe used, such as X-VIVO™ medium, such that the GSK-3 inhibitor and themTOR inhibitor may be added to the commercially available HSC culturingmedium in effective amounts as contemplated and describedhereinthroughout.

As explained above, isolated HSC are cultured in a culture system whichcomprises a culture medium containing the GSK-3 inhibitor and the mTORinhibitor. Such a culture system is suitable for achieving an expansion,such as about a 1-fold expansion, a 2-fold expansion, a 3-foldexpansion, a 4-fold expansion, a 5-fold expansion, a 10-fold expansion,a 20-fold expansion, a 50-fold expansion, a 100-fold expansion, a150-fold expansion, or a 200-fold expansion or more and any and allwhole or partial increments there between of the HSCs. The expanded HSCsmay retain their capability for multilineage differentiation uponintroduction into the body of a patient, preferably a human patient.

HSCs can be cultured in the presence of the at least one GSK-3 inhibitorand at least one mTOR inhibitor for approximately 7 days. Alternatively,the HSCs can be cultured in the presence of at least one GSK-3 inhibitorand at least one mTOR inhibitor for approximately 14 days, approximately28 days, approximately 60 days or approximately 120 days or more, andany and all whole and partial increments there between.

GSK-3 and mTOR Inhibitors

GSK-3 is involved in many cellular and physiological events, includingWnt and Hedgehog signaling, transcription, insulin action, cell-divisioncycle, response to DNA damage, cell death, cell survival, patterning andaxial orientation during development, differentiation, neuronalfunctions, circadian rhythm and others. In humans, two genes, which mapto 19q13.2 and 3q13.3, encode two distinct but closely related GSK-3forms, GSK-3a (51 kDa) and GSK-3b (47 kDa).

GSK-3 is regulated at multiple levels. First, GSK-3b is regulated bypost-translational phosphorylation of Ser9 (inhibitory) and Tyr216(activating) (Ser21 and Tyr279, respectively, in GSK-3α). PhosphorylatedSer9 in the N-terminal domain of GSK-3b acts as a pseudo-substrate thatblocks the access of substrates to the catalytic site. UnphosphorylatedTyr216 in the T-loop domain prevents access of substrates to thecatalytic site, and phosphorylation releases this inhibition. Second,GSK-3b is regulated by interactions with many other proteins. Axin andpresenilin act as docking proteins that allow the substrates to makecontact with the priming kinase [casein kinase (CK1) and protein kinaseA, respectively] and GSK-3. Docking proteins might, thus, specifydifferent GSK-3 functions in the cell. Third, GSK-3 action requires thepriming phosphorylation of its substrates by another kinase on a serineresidue located four amino acids C-terminal to the GSK-3 phosphorylationsite. Fourth, GSK-3 is regulated through its intracellular distribution.

In one exemplary embodiment, lithium, such as in the form of LiCl, maybe used as an inhibitor of GSK-3. As contemplated herein, any otherGSK-3 inhibitor in any form may be used, either separately or incombination, as would be understood by those skilled in the art,provided such inhibitors do not orientate the differentiation of theHSCs into well-defined, committed cell lineages, unless such a result isdesired. Other such GSK-3 inhibitors may include, without limitation,6BIO, CHIR-911, DW21, AR-A014418, TDZD and their related compounds.

mTOR is a kinase protein predominantly found in the cytoplasm of thecell. It acts as a central regulator of many biological processes thatare essential for cell proliferation, angiogenesis, and cell metabolism.mTOR exerts its effects primarily by turning on the cell's translationalmachinery, and is therefore responsible for protein synthesis. mTOR is akey intracellular point of convergence for a number of cellularsignaling pathways.

In one exemplary embodiment, rapamycin, which is also known assirolimus, may be used as an mTOR inhibitor. To inhibit mTOR, rapamycinbinds to an abundant intracellular binding protein, FKBP-12, and thedrug:FKPB-12 complex binds at the rapamycin binding domain. In anotherembodiment, temsirolimus, which is also known as CCI-779, may be used asan mTOR inhibitor. In another embodiment, everolimus, which is alsoknown as RAD001, may be used as an mTOR inhibitor. Everolimus andtemsirolimus are considered rapamycin derivatives, the primarydifference being their pharmacokinetic and pharmacologic properties.Temsirolimus was created by adding an ester to a rapamycin carbon, andadding an ether created everolimus. As contemplated herein, any othermTOR inhibitor in any form may be used, either separately or incombination, as would be understood by those skilled in the art.

As contemplated herein, an effective amount of each inhibitor is addedto the culturing medium. The actual amount of inhibitor added to theculturing medium can vary according to the culturing volume, additionalculturing components, and the particular inhibitor selected. Forexample, about 5 mM LiCl and about 5 nM rapamycin may be an effectiveamount of each inhibitor, and added to X-VIVO15 medium for culturing inmulti-well plates.

Additional Culturing Components

In an alternative embodiment of the present invention, the culturemedium may include at least one cytokine. However, it should beappreciated that the present invention does not require the inclusion ofa cytokine, and thus the addition of at least one cytokine is optional.Without limitation, such cytokines may include interleukins 3 and 6(11-3 and 11-6), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), Flt-3 ligand (FL), and thrombopoietin(TPO). A single cytokine can be added or a combination of two or morecytokines can be added to the culture system. For example, the mediumcan include 11-3 or 11-6, or a combination thereof, or it may includeTPO or CSF or a combination thereof. It is believed that the addition ofat least one cytokine can enhance the expansion of HSC by at least about50%, preferably at least about 100%.

In another alternative embodiment of the present invention, the culturemedium may include at least one stem cell expansion promoting factorwhich is distinct from the aforementioned cytokines. For example, HSCscan be maintained and expanded ex vivo in the presence of stromal cellmedium containing such promotion factors, such as a culture mediumcollected from cultured murine stromal cells where the stromal cellswere cultured in the presence of a leukemia inhibitory factor.

Characterization

At any time point during the culturing of the cells with at least oneGSK-3 inhibitor and at least one mTOR inhibitor, the cells can beharvested and collected for immediate experimental or therapeutic use,or cryopreserved for use at a later time. HSCs, as described herein, maybe cryopreserved according to routine procedures. Preferably, about oneto ten million cells are cryopreserved in medium with about 10% DMSO invapor phase of Liquid N₂. Frozen cells can be thawed by swirling in a37° C. bath, resuspended in fresh proliferation medium, and grown asusual. Cryopreservation is a procedure common in the art and as usedherein encompasses all procedures currently used to cryopreserve cellsfor future analysis and use.

In another aspect, the cells can be harvested and subjected to flowcytometry to evaluate cell surface markers to assess the change inphenotype of the cells in view of the culture conditions. HSCs may becharacterized using any one of numerous methods in the art and methodsdisclosed herein. The cells may be characterized by the identificationof surface and intracellular proteins, genes, and/or other markersindicative of the cells.

Genetic Modification

The cells of the present invention can also be used to express a foreignprotein or molecule for a therapeutic purpose or for a method oftracking their integration and differentiation in a patient's tissue.Thus, the invention encompasses expression vectors and methods for theintroduction of exogenous DNA into the cells with concomitant expressionof the exogenous DNA in the cells such as those described, for example,in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York).

The isolated nucleic acid can encode a molecule used to track themigration, integration, and survival of HSCs once they are placed in thepatient, or they can be used to express a protein that is mutated,deficient, or otherwise dysfunctional in the patient. Proteins fortracking can include, but are not limited to green fluorescent protein(GFP), any of the other fluorescent proteins (i.e., enhanced green,cyan, yellow, blue and red fluorescent proteins; Clontech, Palo Alto,Calif.), or other tag proteins (i.e., LacZ, FLAG-tag, Myc, His6, and thelike) disclosed elsewhere herein.

The present invention is also useful for obtaining HSCs that express anexogenous gene, so that the HSCs can be used, for example, for celltherapy or gene therapy. That is, the present invention allows for theproduction of large numbers of HSCs that express an exogenous gene. Theexogenous gene can, for example, be an exogenous version of anendogenous gene (i.e., a wild type version of the same gene can be usedto replace a defective allele comprising a mutation). The exogenous geneis usually, but not necessarily, covalently linked with (i.e., “fusedwith”) one or more additional genes. Exemplary “additional” genesinclude a gene used for “positive” selection to select cells that haveincorporated the exogenous gene, and a gene used for “negative”selection to select cells that have incorporated the exogenous gene intothe same chromosomal locus as the endogenous gene or both.

An HSC expressing a desired exogenous gene can be used to provide theproduct of the exogenous gene to a cell, tissue, or whole mammal where ahigher level of the gene product can be useful to treat or alleviate adisease, disorder or condition associated with abnormal expression,and/or activity. Therefore, the invention includes an HSC expressing anexogenous gene where increasing expression, protein level, and/oractivity of the desired gene product can be useful to treat or alleviatea disease, disorder or condition.

According to the present invention, gene constructs which comprisenucleotide sequences that encode heterologous proteins are introducedinto the HSCs. That is, the cells are genetically modified to introducea gene whose expression has therapeutic effect in the individual.According to some aspects of the invention, HSCs from the individual tobe treated or from another individual, or from a non-human animal, maybe genetically modified to replace a defective gene and/or to introducea gene whose expression has therapeutic effect in the individual beingtreated.

The term “genetic modification” as used herein refers to the stable ortransient alteration of the genotype of an HSC by intentionalintroduction of exogenous DNA. DNA may be synthetic, or naturallyderived, and may contain genes, portions of genes, or other useful DNAsequences. The term “genetic modification” as used herein is not meantto include naturally occurring alterations such as that which occursthrough natural viral activity, natural genetic recombination, or thelike.

Exogenous DNA may be introduced to an NSC using viral vectors(retrovirus, modified herpes viral, herpes-viral, adenovirus,adeno-associated virus, lentiviral, and the like) or by direct DNAtransfection (lipofection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like). The genetically modified cells of thepresent invention possess the added advantage of having the capacity toproduce differentiated cells in a reproducible fashion using a number ofdifferentiation protocols.

In all cases in which a gene construct is transfected into a cell, theheterologous gene is operably linked to regulatory sequences required toachieve expression of the gene in the cell. Such regulatory sequencestypically include a promoter and a polyadenylation signal.

The gene construct is preferably provided as an expression vector thatincludes the coding sequence for a heterologous protein operably linkedto essential regulatory sequences such that when the vector istransfected into the cell, the coding sequence will be expressed by thecell. The coding sequence is operably linked to the regulatory elementsnecessary for expression of that sequence in the cells. The nucleotidesequence that encodes the protein may be cDNA, genomic DNA, synthesizedDNA or a hybrid thereof or an RNA molecule such as mRNA.

The gene construct includes the nucleotide sequence encoding thebeneficial protein operably linked to the regulatory elements and mayremain present in the cell as a functioning cytoplasmic molecule, afunctioning episomal molecule or it may integrate into the cell'schromosomal DNA. Exogenous genetic material may be introduced into cellswhere it remains as separate genetic material in the form of a plasmid.Alternatively, linear DNA which can integrate into the chromosome may beintroduced into the cell. When introducing DNA into the cell, reagentswhich promote DNA integration into chromosomes may be added. DNAsequences which are useful to promote integration may also be includedin the DNA molecule. Alternatively, RNA may be introduced into the cell.

The regulatory elements for gene expression include: a promoter, aninitiation codon, a stop codon, and a polyadenylation signal. It ispreferred that these elements be operable in the cells of the presentinvention. Moreover, it is preferred that these elements be operablylinked to the nucleotide sequence that encodes the protein such that thenucleotide sequence can be expressed in the cells and thus the proteincan be produced. Initiation codons and stop codons are generallyconsidered to be part of a nucleotide sequence that encodes the protein.However, it is preferred that these elements are functional in thecells. Similarly, promoters and polyadenylation signals used must befunctional within the cells of the present invention. Examples ofpromoters useful to practice the present invention include but are notlimited to promoters that are active in many cells such as thecytomegalovirus promoter, SV40 promoters and retroviral promoters. Otherexamples of promoters useful to practice the present invention includebut are not limited to tissue-specific promoters, i.e. promoters thatfunction in some tissues but not in others; also, promoters of genesnormally expressed in the cells with or without specific or generalenhancer sequences. In some embodiments, promoters are used whichconstitutively express genes in the cells with or without enhancersequences. Enhancer sequences are provided in such embodiments whenappropriate or desirable.

The cells of the present invention can be transfected using well knowntechniques readily available to those having ordinary skill in the art.Exogenous genes may be introduced into the cells using standard methodswhere the cell expresses the protein encoded by the gene. In someembodiments, cells are transfected by calcium phosphate precipitationtransfection, DEAE dextran transfection, electroporation,microinjection, liposome-mediated transfer, chemical-mediated transfer,ligand mediated transfer or recombinant viral vector transfer.

In some embodiments, recombinant viral vectors are used to introduce DNAwith desired sequences into the cell. In some embodiments, recombinantretrovirus vectors are used to introduce DNA with desired sequences intothe cells. In some embodiments, standard calcium phosphate, DEAE dextranor lipid carrier mediated transfection techniques are employed toincorporate desired DNA into dividing cells. Standard antibioticresistance selection techniques can be used to identify and selecttransfected cells. In some embodiments, DNA is introduced directly intocells by microinjection. Similarly, well-known electroporation orparticle bombardment techniques can be used to introduce foreign DNAinto the cells. A second gene is usually co-transfected or linked to thetherapeutic gene. The second gene is frequently a selectableantibiotic-resistance gene. Transfected cells can be selected by growingthe cells in an antibiotic that will kill cells that do not take up theselectable gene. In most cases where the two genes are unlinked andco-transfected, the cells that survive the antibiotic treatment haveboth genes in them and express both of them.

Methods and Uses of HSCs

Isolated HSCs are useful in a variety of ways. These cells can be usedto reconstitute cells in a mammal whose cells have been lost throughdisease or injury. Genetic diseases may be treated by geneticmodification of autologous or allogeneic HSCs to correct a geneticdefect or to protect against disease. Diseases related to the lack of aparticular secreted product such as a hormone, an enzyme, a growthfactor, or the like may also be treated using HSCs. HSCs isolated andcultured as described herein can be used as a source of progenitor cellsand committed cells to treat selected diseases as would be understood bythose skilled in the art, including such diseases and/or injuries wherethe replacement of tissue by the cells of the present invention canresult in a treatment or alleviation of the disease and/or injuries.HSCs cultured or expanded as described herein can be used, as cultured,or they can be used following differentiation into selected cell types,to treat a variety of disorders known in the art to be treatable usingHSCs. The HSCs that are useful in these treatment methods include thosethat have, and those that do not have, an exogenous gene insertedtherein.

The present invention encompasses methods for administering the cells ofthe present invention to an animal, including humans, in order to treatdiseases where the introduction of new, undamaged cells will providesome form of therapeutic relief.

The cells of the present invention can be administered as HSCs or HSCsthat have been induced to differentiate to exhibit at least onecharacteristic of the targeted cell type. The skilled artisan willreadily understand that HSCs can be administered to a recipient as anundifferentiated cell and upon receiving signals and cues from thesurrounding milieu, can differentiate into a desired cell type dictatedby the neighboring cellular milieu. Additionally, HSCs can beadministered as a purified population of cells, or as a heterogeneousmixture of cells, that contains HSCs as the active agent or cell type.

The cells can be prepared for grafting to ensure long term survival inthe in vivo environment. For example, cells are propagated in a suitableculture medium for growth and maintenance of the cells and are allowedto grow to confluency. Any osmotically balanced solution which isphysiologically compatible with the host subject may be used to suspendand inject the donor cells into the host. Formulations of apharmaceutical composition suitable for parenteral administrationcomprise the active ingredient, i.e. the cells, combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents.

The invention also encompasses grafting HSCs (or differentiated HSCs) incombination with other therapeutic procedures to treat a disease ortrauma. Thus, the cells of the invention may be co-grafted with othercells, both genetically modified or non-genetically modified cells whichexert beneficial effects on the patient. Therefore the methods disclosedherein can be combined with other therapeutic procedures as would beunderstood by one skilled in the art once armed with the teachingsprovided herein.

The mode of administration of the cells of the invention may varydepending on several factors including the type of disease beingtreated, the age of the mammal, whether the cells are differentiated ornot, whether the cells have heterologous DNA introduced therein, and thelike. Cells may be introduced to the desired site by direct injection,or by any other means used in the art for the introduction of compounds.The cells can be administered into a host in a wide variety of ways.Modes of administration include, but are not limited to, intravascular,intracerebral, parenteral, intraperitoneal, intravenous, epidural,intraspinal, intrastemal, intra-articular, intra-synovial, intrathecal,intra-arterial, intracardiac, or intramuscular.

Transplantation of the cells of the present invention can beaccomplished using techniques well known in the art as well as thosedescribed herein or as developed in the future. The present inventioncomprises a method for transplanting, grafting, infusing, or otherwiseintroducing HSCs into a mammal, preferably, a human.

In order to transplant the cells of the present invention into a human,the cells are prepared as described herein. Preferably, the cells arefrom the patient for which the cells are being transplanted into(autologous transplantation).

As contemplated herein, the present invention further provides formethods for use in various therapeutic interventions. For example, themethods of the present invention may be used in the treatment of bonemarrow transplant donors and for recipients clinically by providing toeither donors or recipients FDA approved medications that inhibit GSK-3(such as lithium salts or other compounds that contain lithium) and mTOR(such as rapamycin) to expand HSCs and accelerate recovery from bonemarrow suppression. In another example, the present invention provides amethod for expansion of HSCs in culture using GS K-3 inhibitors and mTORinhibitors. As demonstrated herein, inhibition of GSK-3 will activatedownstream Wnt signaling in HSCs and will promote expansion of HSCs. Asdemonstrated herein, this can be a complex using FDA approvedmedications, such as lithium salts, which are already widely usedclinically and are relatively safe. Applications apply to stem cells andother tissues, for example the skin and hair follicles (regeneration ofskin and hair, burns, etc.), colon (regeneration of colonic epithelium,for example, after chemotherapy), and central nervous system (neuronalregeneration for neurodegenerative disorders). Further, the presentinvention may provide for ex vivo expansion of HSCs and improvetransplantation, such as bone marrow transplantation, by expandingeither short-term or long-term HSC's, or both, and improving initialsurvival, such as during the marrow suppression/peripheral cytopeniaphase of BMT. Furthermore, these interventions make it possible toexpand HSC's in human cord blood to improve the success of cord bloodtransplants. This approach can be more generally applicable to the exvivo culture of human stem cells from somatic tissues, human embryonicstem cells, and induced pluripotent stem cells.

In one embodiment of the present invention, the method includesharvesting and culturing a patient's HSCs in culture medium containingat least one GSK-3 inhibitor and at least one mTOR inhibitor fortreatment of cancers of the blood, such as leukemia and lymphoma, wherethe patient's cancerous hematopoietic cells were destroyed via radiationor chemotherapy and stem cells are replaced by stem cell transplantation(SCT). The method includes replacing the cultured HSCs via a bone marrowtransplant. As used herein, the term “stem cell transplant” encompassesall forms of HSC transplant, including bone marrow, peripheral blood,and cord blood HSCs. In another embodiment, the method includes thetransplanting of HSCs collected from the peripheral circulation of amatched donor. In another embodiment, the method includes thetransplanting of HSCs collected from human umbilical cord blood (hUCB).As contemplated herein, and without limitation, the cultured HSCs may beused to treat cancers of the blood such as acute lymphoblastic leukemia,acute myeloblastic leukemia, chronic myelogenous leukemia (CML),Hodgkin's disease, multiple myeloma, and non-Hodgkin's lymphoma andneoplastic diseases of the blood including myelodysplasia andmyeloproliferative disease.

In another embodiment, the aforementioned methods may be used in thetreatment of bone marrow failure syndromes including aplastic anemia,whether acquired or inherited, and paroxysmal nocturnal hemoglobinuria.

In another embodiment, the aforementioned methods may be used in thetreatment of hereditary blood disorders, such as different types ofinherited anemia (failure to produce blood cells), and inborn errors ofmetabolism (genetic disorders characterized by defects in key enzymesneed to produce essential body components or degrade chemicalbyproducts). Without limitation, such blood disorders treatable by themethods of the present invention may include aplastic anemia,beta-thalassemia, Blackfan-Diamond syndrome, globoid cellleukodystrophy, sickle-cell anemia, severe combined immunodeficiency,X-linked lymphoproliferative syndrome, and Wiskott-Aldrich syndrome.Without limitation, inborn errors of metabolism that are treatable withthe methods of the present invention include Hunter's syndrome, Hurler'ssyndrome, Lesch Nyhan syndrome, and osteopetrosis. Because bone marrowtransplantation has carried a significant risk of death, this is usuallya treatment of last resort for otherwise fatal diseases.

As chemotherapeutic techniques aimed at rapidly dividing cancer cellsinevitably hit rapidly dividing hematopoietic cells, the methods of thepresent invention may also be used in treating cancer patients with anautologous stem cell transplant to replace the cells destroyed bychemotherapy. The method may include mobilizing HSCs, collecting themfrom peripheral blood, and culturing the cells in culturing mediumcontaining at least one GSK-3 inhibitor and at least one mTOR inhibitor.The cells may then be stored, such as via cryopreservation, while thepatient undergoes intensive chemotherapy or radiotherapy to destroy thecancer cells. Once the chemotherapy or radiotherapy is completed, thepatient may receive a transfusion of his or her stored HSCs.

In yet another embodiment, the methods of the present invention may beused for treating otherwise untreatable tumors, such as for thetreatment of metastatic kidney cancer or solid tumors that resiststandard therapy, including cancer of the lung, prostate, ovary, colon,esophagus, liver, and pancreas. For example, the method may include anallogeneic stem cell transplant from an HLA-matched sibling whose HSCsare collected peripherally and cultured in a culturing medium containingat least one GSK-3 inhibitor and at least one mTOR inhibitor. The methodincludes transfusing the donor's cultured HSCs into the patient,followed by monitoring the patient's immune cells, such as via DNAfingerprinting, to follow the engraftment of the donor's cells andregrowth of the patient's own blood cells.

In other embodiments, the aforementioned methods may be used fortreating other diseases, such as diabetes, rheumatoid arthritis, systemlupus erythematosis and other autoimmune diseases. For example, HSCscultured in the presence of at least one GSK-3 inhibitor and at leastone mTOR inhibitor may be genetically modified for delivery of genes torepair damaged cells. As contemplated herein, the methods of the presentinvention may be used for treating any disease, disorder or condition inwhich HSCs are used.

Kits

The present invention further includes various kits which comprise aculturing medium, including at least one GSK-3 inhibitor and at leastone mTOR inhibitor, and instructional materials which describe use ofthe culturing medium to perform the methods of the invention. Althoughmodel kits are described below, the contents of other useful kits willbe apparent to the skilled artisan in light of the present disclosure.Each of these kits is contemplated within the present invention.

In one aspect, the invention includes a kit for maintaining andexpanding HSCs ex vivo. The kit is used in the same manner as themethods disclosed herein for the present invention. Briefly, the kit maybe used to maintain and expand freshly harvested HSCs or cryopreservedHSCs in culture. The kit may further include cyropreserved HSCs. Thecryopreserved HSCs may be provided in an appropriate amount as set forthelsewhere herein. The kit may further include genetically modified HSCs.The kit may further include at least one cytokine or a promotion factor.Additionally, the kit comprises instructional material for the use ofthe kit. These instructions simply embody any of the examples andembodiments provided herein.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teachings providedherein.

The materials and methods employed in the experiments and examplesdisclosed herein are now described.

Mice: C57BL/6 WT (CD45.2), CD45.1 congenic (The Jackson Laboratory), andMx-cre;β-catenin^(fl/fl) mice were bred in house in a pathogen-freemouse facility of the University of Pennsylvania. Transplant recipientswere female mice 10-12 weeks old. All work with mice was done accordingto a protocol reviewed and approved by the Institutional Animal Care andUse Committee at the University of Pennsylvania.

FCM and HSC isolation: BM cells were flushed from the long bones (tibiasand femurs) of mice with Hank's buffered salt solution without calciumor magnesium, supplemented with 2% heat-inactivated calf serum. Fordetection of LSK Flk2 CD34 cells, whole BM cells were incubated withBiotin-conjugated monoclonal antibodies to lineage markers, includingB220 (6B2), CD4 (GK1.5), CD8 (53-6.7), Gr-1 (8C5), Mac-1 (M1/70),Ter119, and IL-7R (A7R34), in addition to PE Cy5.5—conjugated anti-Sca-1(Ly6A/E; D7), allophycocyanin—Alexa Fluor 750—conjugated anti-c-kit(ACK2), PE-conjugated anti-Flk2 (Ly-72/A2F10), and Alexa Fluor647—conjugated anti-CD34. Biotin-conjugated lineage markers weredetected using streptavidin-conjugated PE-Texas Red. Nonviable cellswere excluded from sorting and analyses using the viability dye DAPI (1g/ml). Cells were sorted with a FACSAria (BD) or MoFlo (Cytomation)automated cell sorter. Analysis was performed on LSR II or FACSCaliburflow cytometer (BD). Data were analyzed using FlowJo software (TreeStar).

Constructs and lentiviruses: shRNAs sequences were designed usingsoftware from the Broad Institute(http://www.broad.mit.edu/genome_bio/trc/publicSearchForHairpinsForm.php),which identified 5 potential shRNA sequences in Gsk-3 and PTEN. TheshRNAs were cloned into the H1UG1 lentivirus (Balint, et al., 2005, J.Clin. Invest. 115:3166-3176), a 4-component, replication-incompetentsystem derived from FG12 (Qin, et al., 2003, Proc. Natl. Acad. Sci.U.S.A. 100:183-188), which uses the human U1 promoter to drive shRNAexpression and the human Ubiquitin-C promoter to drive expression of GFPfor lineage tracing. H1UG1 was provided by A. Gewirtz (University ofPennsylvania School of Medicine). High-titer lentiviral supernatant wasproduced by transient transfection of 293T cells and was tested inNIH3T3 cells.

BM transduction and transplantation: Lentiviral transduction of5-FU—treated BM cells and transplantation into lethally irradiated (10Gy) recipients was performed as described previously (Pui, et al., 1999,Immunity. 11:299-308).

Cell cycle analysis and BrdU incorporation: Sorted GFP⁺ LSK and GFP⁺ LSKFlk2⁻ cells from primary recipients of Gsk3-rnai or vector control for 4months were incubated, as described previously (Bersenev, et al., 2008,J. Clin Invest. 118:2832-2844), with 5 μg/ml Hoechst 33342 (Invitrogen)in HBSS containing 20 mM HEPES, 5 mM glucose, and 10% FBS at 37° C. for45 minutes, then incubated for 45 minutes with 1 μg/ml Pyronin(Sigma-Aldrich), and analyzed with an LSRII flow cytometer (BDBiosciences). For BrdU labeling, primary recipients of Gsk3-rnai orvector control were fed 0.5 mg/ml BrdU in the drinking water for thelast 7 days of a 4-month transplant, and GFP⁺ LSK Flk2⁺ and GFP⁺ LSKFlk2⁻ cells were sorted. BrdU incorporation was determined by FACSanalysis using APC-conjugated antibodies specific to BrdU and PIaccording to the manufacturer's protocol (BD Biosciences).

Long-term noncompetitive repopulation assay: Adult recipient mice wereirradiated with a Cs-137 Irradiator in 2 equal doses of 5 Gy separatedby at least 2 hours. Cells were injected into the retroorbital venoussinus of anesthetized recipients. Each secondary recipient mousereceived 2×10⁵ sorted GFP⁺BM cells from primary recipients of Gsk3-rnaior vector control, and each tertiary recipient mouse received 4×10⁵sorted GFP⁺BM cells from secondary recipients. Beginning 4 weeks aftertransplantation and continuing for at least 16 weeks, blood wascollected from the tail veins of recipient mice, red blood cells werelysed by ammonium chloride-potassium (Ack) buffer, and the remainingcells were stained with directly conjugated antibodies to B220 (6B2),Mac-1 (M1/70), CD4 (L3T4), CD8 (Ly-3), and Gr-1 (8C5) to monitorengraftment by FCM.

Long-term competitive repopulation assay: Sorted GFP⁺ cells from primaryrecipients of Gsk3-rnai or vector control (tester) were transplantedinto lethally irradiated B6 recipients together with 2×10⁵ competitor B6BM cells (CD45.2⁺; Bersenev, et al., 2008, J. Clin Invest.118:2832-2844; Szilvassy, et al., 1990, Proc. Natl. Acad. Sci. U.S.A87:8736-8740; Maillard, et al., 2008, Cell Stem Cell. 2:356-366). Inlimiting dilution analyses, decreasing numbers of tester GFP⁺ cells wereused (i.e., 1×10⁶, 5×10⁵, 1×10⁵, and 2×10⁴). Beginning 4 weeks aftertransplantation and up to 16 weeks, blood was collected from the tailveins of recipient mice, red blood cells were lysed in Ack buffer, andthe remaining cells were stained with directly conjugated antibodies toB220 (6B2), Mac-1 (M1/70), CD4 (L3T4), CD8 (Ly-3), and Gr-1 (8C5) tomonitor engraftment by FCM. Blood and BM were analyzed 16 weeks aftertransplantation. The number of competitive repopulation units wascalculated with L-Calc software (StemCell Technologies; Bersenev, etal., 2008, J. Clin Invest. 118:2832-2844; Maillard et al., 2008, CellStem Cell. 2:356-366).

Methylcellulose culture: Sorted GFP⁺BM cells were plated in individualwells of 6-well plates (Corning) containing 550 μl 1.0% methylcellulose(Stem Cell Technologies) as previously described (Trowbridge, et al.,2006, Nature. 441:518-522). The methylcellulose was supplemented with 1%penicillin/streptomycin (Gibco; Invitrogen), 50 ng/ml SCF, 10 ng/mlIL-3, 10 ng/ml IL-6, and 3 U/ml erythropoietin. Colonies were incubatedat 37° C. in humidified incubators at 5% CO₂. Colony formation wasscored by counting all colonies of greater than 30 viable cells after10-14 days of culture.

Administration of polyI:polyC and rapamycin: As described previously(Cobas, et al., 2004, J. Exp. Med. 199:221-229), polyI:polyC(Sigma-Aldrich) was resuspended in Dulbecco PBS at 2 mg/ml. Micereceived 25 μg/g polyI:polyC every other day for 2 weeks. Rapamycin (LCLaboratories) was dissolved in absolute ethanol at 10 mg/ml and dilutedin 5% Tween-80 (Sigma-Aldrich) and 5% PEG-400 (Hampton Research) beforeinjection and was administered by intraperitoneal injection at 4 mg/kgrapamycin in 200 μl total volume/injection every other day for 8 weeks.

Statistics: All data are mean±SD. Statistical significance wasdetermined by a 2-tailed Student's t test, and a P value less than 0.05was considered significant.

The results of the experiments presented herein are now described.

Example 1: Lithium Increases Hematopoietic Stem/Progenitor Cells

Lithium increases circulating CD34+ stem cells (Bailin, et al., 1998, BrJ Haematol 100:219-221) in humans, increases neutrophil count in a highpercentage of treated patients, and may also stimulate other lineages(Shopsin, et al., 1971, Clin Pharmacol Ther 12:923-928; Boggs, et al.,1983, Seminars in Hematology 20:129-138; Barr, et al., 1983, CanadianMedical Association Journal 128:123-126; Tisman, et al. 1972, BritishJournal of Haematology 24:767-771; Joyce, 1984, British Journal ofHaematology 56:307-321; Bille, et al., 1975, Acta Medica Scandinavica198:281-286; Ricci, et al., 1981, Haematologica 66:627-633; Focosi, etal., 2009, Journal of Leukocyte Biology 85:20-28). In rodents, lithiumincreases peripheral blood counts and enhances stem and progenitor cellnumbers in ex vivo and in vivo assays. These studies led to thehypothesis that the effects of lithium are mediated at the level of theHSC and/or HPC (Joyce, 1984, British Journal of Haematology 56:307-321;Gallicchio et al., 1992, Journal of Medicine 23:195-216; Gallicchio, etal., 1980, Blood 56:1150-1152). With immunophenotypic markers to detectdiverse hematopoietic cell types, the effect of lithium has beenreexamined on hematopoiesis in C57/B6 mice using FCM.

Mice received dietary LiCl (or NaCl as control) at a dose that achievesa serum lithium concentration of 1.0 mEq/L (O'Brien, et al., 2004,Journal of Neuroscience 24:6791-6798), similar to therapeuticconcentrations in bipolar disorder patients (there was no change in theoverall well being of the animals after two-three weeks on lithium).After 2 weeks, bone marrow was isolated and cells were analyzed by FCM.Lithium caused a significant increase in the number of LSK cellscompared with NaCl treated animals, as depicted in FIG. 1A, consistentwith an increase in HSCs and HPCs, and a doubling of the overall marrowcellularity, as depicted in FIG. 1B. To confirm this, the expression ofthe SLAM family receptors CD150, CD48, and CD244 was examined (Kiel, etal., 2005, Cell 121:1109-1121) and it was observed that there was anapproximately 2.3-fold increase in the number of CD150+CD48−CD244−cells, an immunophenotypic population highly enriched for HSCs (data notshown). Histological analysis of bone marrow morphology showed nosignificant differences in maturity or cellular morphology inlithium-treated versus control marrows, as reported previously (notshown). Consistent with published observations in humans and rodents,the percentage of neutrophils in peripheral blood also increased 30-40%in lithium-treated mice (data not shown). Inclusion of CD34 and Flk2 inFCM analysis shows that lithium primarily increases theCD34+Flk2-population, consistent with an increase in ST-HSCs, alsodepicted in FIG. 1A.

Example 2: GSK-3 as the Target of Lithium in HSC/HPCs

Inhibition of GSK-3 provides a compelling explanation for many of theknown effects of lithium (Klein, et al., 1996, Proc. Nat'l. Acad. Sci.U.S.A. 93:8455-8459), but lithium also inhibits inositol monophosphataseand structurally related phosphomonoesterases, some of which are highlysensitive to lithium (Gurvich et al., 2002, Pharmacol Ther 96:45-66). Totest further whether inhibition of GSK-3 explains the hematopoieticeffects of lithium, selective GSK-3 inhibitors were used that areunlikely to have off-target effects that overlap with lithium (Bain, etal., 2003, Biochem J 371 (Pt. 1):199-204; Meijer, et al., 2003, ChemBiol 10:1255-1266; Williams, et al., 2005, Angew Chem Int Ed Engl.44:1984-1987). The selective GSK-3 inhibitor, 6-bromo-indirubin 3′-oxime(6BIO) has an IC50 for GSK-3 in the nanomolar range (Meijer, et al.,2003, Chem Biol 10:1255-1266). 6BIO caused a pronounced increase in thenumber of LSK cells after a 2-week treatment, as depicted in FIG. 1C.6BIO also increased marrow cellularity, similar to lithium. Theseobservations are consistent with Trowbridge et al, who observed anapproximately 50% increase in LSK cells in mice treated with the GSK-3inhibitor CHIR-911 (Trowbridge, et al., 2006, Nat Med 12:89-98).Goessling et al also reported that 6BIO increases HSCs in mice asmeasured by long-term competitive repopulation assay (Goessling, et al.,2009, Cell 136:1136-1147), consistent with the observation of increasedLSK cells after 6BIO treatment described herein. Taken together, thesepharmacological data support the hypothesis that lithium increases HSCsthrough inhibition of GSK-3.

To test whether progenitor cells are increased by lithium treatment,colony formation assays were performed. Previous work in the 1980's withlithium-treated rodents demonstrated an increase in colony forming units(CFU) in ex vivo assays and by CFU-S formation in short-term transplants(Joyce, 1984, British Journal of Haematology 56:307-321; Levitt, et al.,1980, New England Journal of Medicine 302:713-719). To confirm thesestudies, bone marrow cells were isolated from lithium treated mice andcultured in methylcellulose with hematopoietic cytokines. Marrow fromlithiumtreated animals showed a two-fold increase in total colonyinitiating cells compared to control, similar to earlier reports (datanot shown) and consistent with more recent observations with smallmolecule GSK-3 inhibitors, which increase CFUs ex vivo and CFU-Sapproximately 1.5 to 2-fold (Trowbridge, et al., 2006, Nat Med 12:89-98;Goessling, et al., 2009, Cell 136:1136-1147).

To test in a side-by-side comparison whether structurally diverse GSK-3inhibitors expand HPCs similar to lithium, c-Kit+ cells were purifiedfrom control mice and treated for three days with GSK-3 inhibitorsincluding lithium, 6BIO (Meijer, et al., 2003, Chem Biol 10:1255-1266;Goessling, et al., 2009, Cell 136:1136-1147), AR-A014418 (Bhat, et al.,2003, Journal of Biological Chemistry 278:45937-45945), and theorganometallic GSK-3 inhibitor DW21 (Williams, et al., 2005, Angew ChemInt Ed Engl. 44:1984-1987). The number of cells after three days wassimilar in each group, as depicted in FIG. 1E. Treated cells were washedand an equal number from each sample was then added to methylcellulosewith hematopoietic cytokines and cultured for 10-14 days. Each of theGSK-3 inhibitors induced a marked increase in hematopoietic colonynumber, as depicted in FIG. 1D, strongly supporting the hypothesis thatlithium expands the HPC population by inhibiting GSK-3 withinhematopoietic cells.

Example 3: Gsk-3 Loss of Function in Hematopoietic Cells

Therapeutic lithium increases the number of circulating CD34⁺ stem cellsin humans (Ballin, et al., 1998, Br. J. Haematol. 100:219-221),increases peripheral blood counts, especially neutrophils, in a highpercentage of treated patients, and enhances stem and progenitor cellnumbers in rodents (Boggs, et al., 1983, Semin. Hematol. 20:129-138;Joyce, 1984, Br. J. Haematol. 56:307-321; Ricci, et al., 1981,Haematologica. 66:627-633; Focosi, et al., 2009, J. Leukoc. Biol.85:20-28; Gallicchio, et al., 1992, J. Med. 23:195-216; Gallicchio, etal., 1980, Blood. 56:1150-1152). However, since these early studies wereperformed, immunophenotypic markers of HSCs and HPCs have becomeavailable (Purton, et al., 2007, Cell Stem Cell. 1:263-270). Flowcytometry (FCM) on BM from lithium-treated mice were used, and anincrease in immunophenotypic HSCs/HPCs was found, as detected by LSKmarkers (Purton, et al., 2007, Cell Stem Cell. 1:263-270; See FIG. 1A)or by detection of the SLAM marker immunophenotype (CD150⁺CD48⁻)characteristic of HSCs (Kiel, et al., 2005, Cell. 121:1109-1121; datanot shown). The selective GSK-3 inhibitor 6-bromo-indirubin 3′-oxime(6BIO) also increased the number of LSK cells after 2 weeks, as depictedin FIG. 1C, consistent with recent reports using 6BIO (Goessling, etal., 2009, Cell. 136:1136-1147) or the GSK-3 inhibitor CHIR-911(Trowbridge, et al., 2006, Nat. Med. 12:89-98). It was also observedherein that a parallel increase in BM cellularity after treatment witheither lithium or 6BIO, with no observable change in BM architecture, asdepicted in FIG. 1B (also data not shown). Lithium, 6BIO, AR-A014418,and other structurally distinct GSK-3 inhibitors also increasedhematopoietic colony formation ex vivo, as depicted in FIG. 1D.

The parallel effects of lithium and alternative GSK-3 inhibitors supportthe hypothesis that GSK-3 is a significant target of lithium inHSCs/HPCs and suggest a critical function for GSK-3 in hematopoiesis.However, results based on systemically delivered inhibitors do notaddress whether GSK-3 functions cell autonomously in HSCs/HPCs; inaddition, off-target effects remain a formal possibility. As Gsk-3 lossof function has not previously been reported in HSCs, depletion of Gsk-3in BM cells was tested using RNAi and conventional Gsk3b KO (Hoeflich,et al., 2000, Nature. 406:86-90). Two distinct shRNAs that targetsequences conserved in both Gsk3a and Gsk3b were cloned into alentivirus that also expresses GFP (Balint, et al., 2005, J. Clin.Invest. 115:3166-3176). Donor BM cells were infected with control orshRNA constructs (Gsk3-rnai-C2 and Gsk3-rnai-C4) and transplanted intolethally irradiated primary recipients. Peripheral blood was sampled at4-week intervals for 20 weeks to confirm engraftment. After 8 weeks, amajority of peripheral blood cells in both Gsk3-rnai and control vectortransplants were derived from GFP⁺ donor cells, including T cells (CD4⁺and CD8⁺), B cells (B220⁺), myeloid cells (Gr1⁺CD11b⁺), and erythroidcells (TER119⁺), indicating successful engraftment and reconstitution,as depicted in FIG. 2A (other data not shown). The contribution to themature myeloid lineage, as marked by Gr1⁺CD11b⁺ cells, was increased at8 and 12 weeks in hosts receiving Gsk3-rnai BM, as depicted in FIG. 2B,similar to known effects of lithium treatment (Boggs et al., 1983,Semin. Hematol. 20:129-138; Joyce, 1984, Br. J. Haematol. 56:307-321;Ricci, et al., 1981, Haematologica. 66:627-633; Focosi, et al., 2009. J.Leukoc. Biol. 85:20-28; Gallicchio, et al., 1992, J. Med. 23:195-216;Gallicchio, et al., 1980, Blood. 56:1150-1152). GSK-3α and GSK-3 proteinlevels remained low in BM harvested from primary recipients 4 monthsafter transplantation with both Gsk3-rnai vectors, but not with controllentivirus, as depicted in FIG. 2C. Furthermore, β-catenin proteinlevels were elevated in Gsk3-depleted BM cells harvested from primaryrecipients of Gsk3-rnai cells, also depicted in FIG. 2C, consistent withreduced GSK-3-dependent phosphorylation and activation of Wnt signaling.Because subsequent results with the Gsk3-rnai-C2 and Gsk3-rnai-C4constructs were similar, only data using the Gsk3-rnai-C2 construct areshown.

Importantly, both percentage and number of immunophenotypic HSCs/HPCsincreased greater than 4-fold in Gsk3-rnai-transduced cells comparedwith control cells, as assessed by the increase in GFP⁺ LSK cells anddepicted in FIGS. 2D-2F, as well as CD150⁺CD48⁻ cells, as depicted inFIG. 2G. Multiparametric FCM analysis of CD34 and flk-2 showed anincrease in immunophenotypic short-term HSCs (ST-HSCs) and long-termHSCs (LT-HSCs; FIG. 2E). The total cellularity of the BM was onlymarginally increased, as depicted in FIG. 3B, in contrast to the effectof the systemically delivered GSK-3 inhibitors (FIG. 1B). Furthermore,BM harvested at 4 months from primary transplants ofGsk3-rnai-transduced BM yielded greater than 4-fold more colonies inmethylcellulose culture than did control BM, as depicted in FIG. 2H.

To explore the mechanism by which Gsk-3 depletion expands the size ofthe phenotypic HSC/HPC pool, the cell cycle and survival status ofGFP-marked LSK cells was examined. GFP⁺ LSK or GFP⁺ LSK Flk2⁻ cells werepurified from control or Gsk3-rnai-transduced BM after 4-5 months inprimary recipients and stained with pyronin and Hoechst followed by FCM(Bersenev, et al., 2008, J. Clin Invest. 118:2832-2844). Compared withcontrols, approximately 2-fold more Gsk3-depleted cells had entered theS/M/G₂ phases of the cell cycle in both the GFP⁺ LSK and GFP⁺ LSK Flk2⁻populations, as depicted in FIG. 4A, demonstrating increased cycling ofGsk3-deficient LSK cells. Transplant recipients were also fed BrdU for 7days prior to BM harvest. Incorporation of BrdU coupled with analysis ofpropidium iodide (PI) staining confirmed that Gsk-3 depletion increasedthe percentage of LSK cells in S/M/G₂ more than 2-fold, as depicted inFIG. 4B. Annexin V staining in Gsk3-deficient LSK cells was notsignificantly different from that of controls (FIG. 3A), indicating thatthe increase in LSK cells is not caused by a change in the rate of celldeath. These data indicate that loss of Gsk-3 results in acceleratedcell cycle progression within the LSK cell population.

Example 4: Functional HSCs are Reduced in Gsk3-Deficient BM

Because inhibition of GSK-3 activity or expression increasedimmunophenotypic HSCs and HPCs and increased functional HPCs within theLSK cell population, competitive repopulating units were measured(Szilvassy, et al., 1990, Proc. Natl. Acad. Sci. U.S.A 87:8736-8740) asa functional test of HSCs in Gsk3-depleted versus control BM. Controland Gsk3-rnai-infected BM was transplanted to irradiated recipients andharvested after 4 months. Sorted GFP⁺ cells were mixed at dilutions from2×10⁴ to 1×10⁶ cells with a constant number (2×10⁵) of GFP⁻ recipientcells and transplanted to lethally irradiated recipient mice. Afteranother 4 months, BM was harvested, and chimerism was analyzed as thepercentage of GFP⁺ cells. Despite the increase in phenotypic HSCs andHPCs observed in primary transplants, Gsk3-deficient cells were lessefficient in competitive reconstitution than were control cells, asdepicted in FIGS. 5B and 5C (see also Table 1, below), withapproximately 3-fold fewer functional HSCs in the Gsk3-rnai group.

TABLE 1 Functional HSCs are reduced by Gsk3-rnai in competitiverepopulation assay No. Donor Cells Vector Control Gsk3-rnai 2 × 10⁴ 1/50/5 1 × 10⁵  9/12  3/12 5 × 10⁵ 10/10 10/10 1 × 10⁶ 10/10 10/10Cohorts of lethally irradiated mice were transplanted with the indicatednumbers of GFP+ vector-control (H1UG) or Gsk3-rnai-transduced bonemarrow cells combined with 2×10⁵ host-derived bone marrow cells. Thepercentage of donor-derived cells in bone marrow was analyzed 16 weeksafter reconstitution; greater than 1% donor-derived cells was consideredpositive engraftment. Values show number of mice with positiveengraftment/total mice transplanted. Data from the cohort of micereceiving 1×10⁶GFP+ cells are also included in FIGS. 5, B and C. Thefrequency of long-term competitive repopulation units was calculatedusing Poisson statistics; vector control reconstitution frequency,1:73,000 (95% confidence interval, 1:54,000-1:100,000); Gsk3-rnaireconstitution frequency, 1:200,000 (95% confidence interval,1:150,000-1:270,000).

As a further test of LT-HSC function, serial, noncompetitivetransplantation to secondary and tertiary lethally irradiated hosts wasperformed. BM was harvested from primary recipients after 4 months, and2×10⁵ sorted GFP⁺ cells were transplanted into lethally irradiatedsecondary recipients. Of recipients of Gsk3-depleted cells, 50% diedwithin 1 month of transplantation, whereas 100% of vector control BMrecipients survived, as depicted in FIG. 6A. All control recipients andsurviving recipients of Gsk3-rnai-infected cells showed long-termmultilineage reconstitution with high levels of GFP⁺ donor cellscontributing to multiple peripheral blood lineages (data not shown) andBM. However, in contrast to primary recipients, BM harvested at 4 monthsfrom surviving Gsk3-rnai secondary recipients did not show an increasein GFP⁺ LSK cells, as depicted in FIG. 6B, or CD150⁺CD48⁻ cells (datanot shown), despite the higher number of GFP⁺ LSK CD150⁺CD48⁻ cellsoriginally present in the Gsk3-rnai donor BM (See FIGS. 2D and 2E).Reduction in GSK-3α and GSK-3β protein was confirmed by Western blot ofGFP⁺BM cells harvested from secondary recipients after 4 months (datanot shown). Analysis of CD34 and flk-2 further showed thatimmunophenotypic LT-HSCs were decreased in Gsk3-rnai secondaryrecipients, and the increase in ST-HSCs (LSK Flk2⁺CD34⁺) was attenuatedapproximately 2-fold, as depicted in FIGS. 6C and 6D, compared with thatin primary recipients (See FIG. 2E). Similarly, colony formation wasreduced 2-fold in secondary recipients of Gsk3-rnai compared with vectorcontrol, as depicted in FIG. 6E. Furthermore, the proportion ofgranulocyte-monocyte progenitor cells in the GFP⁺ population increased,while the percentage of common lymphoid progenitor cells decreasedsignificantly, as depicted in FIG. 6F. These observations suggest thatGsk-3 is required for the maintenance of LT-HSCs and that prolonged lossof GSK-3 activity may promote exit of HSCs from the stem cell pool. Toextend this analysis, GFP⁺ cells recovered from secondary recipients wastransplanted to lethally irradiated tertiary recipients, where 12 of 20mice receiving Gsk3-depleted BM died within 4 months, as depicted inFIG. 6G. LSK cells were reduced 3- to 4-fold in Gsk3-depleted BM insurviving tertiary recipients, as depicted in FIG. 6H, and this markedreduction in HSCs was confirmed by the reduced level of CD150⁺CD48⁻cells (data not shown). These data indicate that loss of Gsk-3 leads toa progressive decline in HSC function and/or number in the course oflong-term serial transplantation. This apparent positive function ofGsk-3 in HSC maintenance was further supported by serial transplantationof HSCs from Gsk3b KO mice (see below).

Example 5: Role of Wnt/β-Catenin Signaling in Response to Gsk-3Knockdown

Gsk-3 loss-of-function mutations result in stabilization of β-cateninprotein and constitutive activation of Wnt signaling (Doble, et al.,2007, Dev. Cell. 12:957-971; Siegfried, et al., 1992, Cell.71:1167-1179). Thus, the effects of GSK-3 inhibitors or Gsk-3 depletionin HSCs/HPCs could be mediated by activation of downstream Wntsignaling. Wnt signaling is active in HSCs under basal conditions(Trowbridge, et al., 2006, Nat. Med. 12:89-98; Goessling, et al., 2009,Cell. 136:1136-1147; Reya, et al., 2003, Nature. 423:409-414; Fleming,et al., 2008, Cell Stem Cell. 2:274-283) and is further activated inHSCs isolated from lithium-treated Bat-gal (Maretto, et al., 2003, Proc.Natl. Acad. Sci. U.S.A 100:3299-3304) reporter mice (data not shown).Furthermore, β-catenin protein levels were persistently elevated inGsk3-rnai-infected BM cells, as depicted in FIG. 2C, which suggests thatdownstream Wnt signaling is activated in these Gsk3-depleted cells.

Although the requirement for Wnt signaling in basal hematopoiesisremains controversial, activation of Wnt signaling can enhance HSC selfrenewal and HPC function in vivo and ex vivo (Trowbridge, et al., 2006,Nat. Med. 12:89-98; Goessling, et al., 2009, Cell. 136:1136-1147;Austin, et al., 1997, Blood. 89:3624-3635; Van Den Berg, et al., 1998,Blood. 92:3189-3202; Willert, et al., 2003, Nature. 423:448-452; Reya,et al., 2003, Nature. 423:409-414; Baba, et al., 2006, J. Immunol.177:2294-2303). It was therefore tested whether β-catenin is requiredfor the effects of Gsk-3 depletion on hematopoiesis using a conditionalβ-catenin loss-of-function allele (β-catenin^(fl/fl) crossed tointerferon-inducible Mx-cre mice (Cobas, et al. 2004, J. Exp. Med.199:221-229). Cre was induced in Mx-cre,β-catenin^(fl/fl) mice withpolyinosine-polycytidine (polyI:polyC), and BM was harvested andinfected with control or Gsk3-rnai lentivirus. After infection, cellswere transplanted to lethally irradiated hosts, and after 4 months, BMfrom these primary recipients was harvested and analyzed by FCM.Depletion of Gsk-3 increased the number of GFP⁺ LSK cells (as well asCD150⁺CD48⁻ cells) derived from WT hosts, and loss of β-catenin blockedthis effect in primary recipients, as depicted in FIG. 7A. In addition,β-catenin^(fl/fl) conditional KO (CKO) blocked the increase in colonyformation observed in Gsk3-rnai;β-catenin^(+/+) BM (compare FIG. 8 andFIG. 2H), demonstrating that β-catenin is required for the effect ofGsk-3 depletion in primary transplanted HSCs/HPCs.

BM from Gsk3-rnai, WT, and β-catenin CKO primary recipients was alsotransplanted to secondary lethally irradiated recipients. As withGsk3-rnai;β-catenin^(+/+) BM, transplantation ofGsk3-rnai;β-catenin^(fl/fl) CKO BM resulted in reduced survival. After 4months, BM was harvested, and LSK cells were measured within the GFP⁺population. Loss of β-catenin resulted in a 3-fold reduction in LSKcells after secondary transplantation of Gsk3-depleted BM, as depictedin FIG. 7B. BM from each group of secondary recipients was alsotransplanted to tertiary recipients, but the survival ofGsk3-rnai;β-catenin^(fl/fl) recipients at 4 months was too low foranalysis (data not shown). The serial BM from WT and β-catenin CKOtransplantation data, also summarized in FIG. 7C, demonstrate thatβ-catenin contributes to HSC maintenance throughout successivetransplants of Gsk3-deficient BM cells. These data, taken together withpreviously published observations from others (Goessling, et al., 2009,Cell. 136:1136-1147; Austin, et al., 1997, Blood. 89:3624-3635; Van DenBerg, et al., 1998, Blood. 92:3189-3202; Willert, et al., 2003, Nature.423:448-452; Reya, et al., 2003, Nature. 423:409-414; Jeannet, et al.,2008, Blood. 111:142-149; Baba, et al., 2006, J. Immunol. 177:2294-2303;Zhao, et al., 2007, Cancer Cell. 12:528-541; Luis, et al., 2009, Blood.113:546-554; Fleming, et al., 2008, Cell Stem Cell. 2:274-283), supporta positive role for the Wnt/β-catenin pathway in HSC maintenance in thecontext of reduced GSK-3 activity, but also suggest that an additionalGSK-3-regulated pathway plays a distinct and possibly antagonistic rolein HSC maintenance.

Example 6: mTOR Inhibition Expands HSCs in Gsk3-Deficient BM

Although GSK-3 regulates multiple pathways, the initial expansion andsubsequent depletion of phenotypic HSCs—as well as the reduction infunctional HSCs—observed with Gsk-3 knockdown was strikingly similar tothe hematopoietic phenotype previously reported for Pten KO mice(Yilmaz, et al., 2006, Nature. 441:475-482; Zhang, et al., 2006, Nature.441:518-522). PTEN is a negative regulator of the PI3K/Akt pathway andmaintains GSK-3 activity by antagonizing Akt-dependent phosphorylationof GSK-3. Pten CKO within HSCs activates the mTOR pathway, and thetransient expansion and subsequent depletion of HSCs in Pten KO cellscan be rescued by treatment of mice with the mTOR inhibitor rapamycin.However, the mechanism for PTEN suppression of mTOR activity has notbeen fully elucidated. GSK-3 was recently shown to inhibit mTORsignaling through phosphorylation of Tsc2, which, together with Tsc1,antagonizes mTOR signaling (Inoki, et al., 2006, Cell. 126:955-968).Furthermore, deletion of Tsc1 increases proliferation and reduces selfrenewal of HSCs, similar to loss of Pten (Chen, et al., 2008, J. Exp.Med. 205:2397-2408; Gan, et al., 2008, Proc. Natl. Acad. Sci. U.S.A.105:19384-19389) and reduced Gsk-3 expression. Thus, it is believed thatthe reduction in HSCs in Gsk3-depleted BM is caused by activation ofmTOR. To test this Western blotting and FCM were used to examinephosphorylation of ribosomal protein S6, a marker for activation ofmTOR, in Gsk3-deficient and control BM. S6 phosphorylation was modestlyincreased in primary recipients of Gsk3-rnai BM (data not shown) andmore demonstrably increased in secondary recipients, as depicted inFIGS. 9A and 9B). S6 phosphorylation was not affected by deletion ofβ-catenin in either primary or secondary recipients.

These observations suggest that GSK-3 is a downstream target ofPTEN-regulated pathways in hematopoietic cells. To test this directly,expression of Pten in BM cells was reduced by lentivirally mediated RNAiand harvested BM from primary hosts after 4 months. The inhibitoryphosphorylation of GSK-3β (serine-9) increased, as depicted in FIG. 6C,which was consistent with reduced PTEN activity and increasedAkt-mediated GSK-3β phosphorylation. Knockdown of Pten in NIH-3T3 cellsalso increased phosphorylation of GSK-3β, as depicted in FIG. 9D. Thesedata indicate that GSK-3β is indeed a downstream target of PI3K/PTENregulation in hematopoietic cells.

Next, secondary recipients of Gsk3-rnai and control BM were treated withrapamycin, as this prevents HSC depletion in Pten KOs (Yilmaz, et al.,2006, Nature. 441:475-482). Rapamycin restored the LSK population insecondary recipients of Gsk3-depleted BM, with a 3- to 4-fold increasein LSK cells, compared with vehicle-injected controls, as depicted inFIGS. 9E and 9F, which suggests that Gsk-3 depletion increases the HSCpopulation above baseline, perhaps through activation of Wnt/β-cateninsignaling in the absence of the antagonistic influence of mTOR signalingin HSCs. Colony formation also increased 2- to 3-fold in BM harvestedfrom rapamycin-treated Gsk3-rnai recipients, as depicted in FIG. 9G,further demonstrating that rapamycin rescues the effect of Gsk3-rnai insecondary recipients. Furthermore, rapamycin treatment also rescuedsurvival of tertiary recipients of Gsk3-depleted BM, as depicted in FIG.6H, which indicates that inhibition of mTOR preserves LT-HSCs inGsk3-depleted BM. However, it will be important in future work tomeasure the number of functional HSCs after treatment with rapamycinusing competitive repopulation assays.

Example 7: Gsk3b is Required for Maintenance of the HSC-Enriched LSKCell Population

Surprisingly, reduced expression of Pten had little effect onphosphorylation of GSK-3α, as depicted in FIG. 9D, raising thepossibility that GSK-3β is a specific target of PI3K/PTEN regulation inHSCs. Although Gsk3a and Gsk3b are structurally similar and are clearlyredundant in Wnt signaling (Doble, et al., 2007, Dev. Cell. 12:957-971),the 2 genes are not redundant in all contexts (Hoeflich, et al., 2000,Nature. 406:86-90; MacAulay, et al., 2007, Cell Metab. 6:329-337). Totest the specific role of Gsk3b in HSC maintenance, transplants wereperformed with hematopoietic cells derived from Gsk3b^(−/−) embryos,which express WT levels of Gsk3a. Homozygous loss of Gsk3b in mice islethal between 15 and 18 days of gestation (Hoeflich, et al., 2000,Nature. 406:86-90). Thus, fetal liver cells were harvested from E17.5WT, Gsk3b^(+/−), and Gsk3b^(−/−) embryos and transplanted into lethallyirradiated adult mice. After 4 months, recipients of WT, Gsk3b^(+/−),and Gsk3b^(−/−) BM showed long-term multilineage reconstitution derivedfrom donor cells, as depicted in FIGS. 10A and 10B. However, there wasno significant increase in the percentage or absolute number of LSK orCD150⁺CD48⁻ cells in primary hosts receiving Gsk3b^(−/−) fetal livercells, as depicted in FIG. 10C. This lack of increase in HSCs in primaryrecipients with selective loss of Gsk3b is in contrast to RNAi-mediatedknockdown of both Gsk3a and Gsk3b. Because the increase in HSCs afterGsk-3 depletion required β-catenin, this observation is consistent withthe well-established redundant roles for Gsk3a and Gsk3b in Wntsignaling (Doble, et al., 2007, Dev. Cell. 12:957-971). However, insecondary transplant recipients, Gsk3b^(−/−) donor cells demonstrated a3- to 4-fold reduction in LSK cells compared with WT cells, as depictedin FIG. 10D, which suggests that Gsk3b regulates HSC homeostasis througha Wnt/β-catenin-independent pathway that becomes evident in secondarytransplants and is not compensated for by Gsk3a. Although the respectiveroles of Gsk3a and Gsk3b may differ in fetal versus adult HSCs, theseobservations, taken together with the selective phosphorylation ofGSK-3β when Pten expression is reduced in hematopoietic cells (or infibroblasts), suggest that GSK-3β is a selective target ofPTEN-regulated pathways and is required for the maintenance of HSC selfrenewal.

As demonstrated herein, Gsk-3 plays an essential role for in themaintenance of LT-HSCs. Gsk-3 loss of function phenocopies Pten and Tsc1mutations in HSCs/HPCs, supporting the belief that GSK-3 functionsdownstream of PTEN to suppress mTOR-dependent HSC activation and lineagecommitment. However, inhibition of GSK-3 also stabilizes β-cateninwithin HSCs/HPCs to induce a β-catenin-dependent increase in phenotypicHSCs, and β-catenin KO accelerates the loss of LT-HSCs in Gsk3-depletedBM, consistent with prior reports with activators of Wnt signaling(Austin, et al., 1997, Blood. 89:3624-3635; Van Den Berg et al., 1998,Blood. 92:3189-3202; Willert et al. 2003, Nature. 423:448-452; Reya, etal., 2003, Nature. 423:409-414; Baba, et al., 2006, J. Immunol.177:2294-2303; Fleming, et al., 2008, Cell Stem Cell. 2:274-283). Thus,as depicted in FIG. 11, Gsk-3 functions in at least two apparentlyopposing processes within HSCs/HPCs. This indicates that Gsk-3 plays anessential role in regulating the balance between self renewal andlineage commitment in HSC, and also supports the belief that the highlyprevalent effects of lithium on hematopoiesis in bipolar patients aremediated by inhibition of GSK-3, and suggest a therapeutic approach,using currently approved GSK-3 and mTOR inhibitors, to expand HSCs invivo.

Pten KO in HSCs/HPCs leads to activation and subsequent depletion ofHSCs, increased lineage commitment resembling myeloproliferativedisorder, and leukemia (Yilmaz et al., 2006, Nature. 441:475-482; Zhang,et al., 2006, Nature. 441:518-522), and this is prevented by the mTORinhibitor rapamycin, which suggests that PTEN-mediated suppression ofmTOR is required for maintenance of quiescent HSCs. Similarly, deletionof the mTOR inhibitor Tsc1 shifts HSCs from a quiescent to aproliferative state and reduces HSC self renewal (Chen, et al., 2008, J.Exp. Med. 205:2397-2408; Gan, et al., 2008, Proc. Natl. Acad. Sci. U.S.A105:19384-19389). As demonstrated herein, reduced expression of Gsk-3,either through RNAi or by homozygous Gsk3b KO, yielded a similarhematopoietic phenotype. This Gsk-3 phenotype was also reversed byrapamycin, and GSK-3β phosphorylation increased in Pten-depleted BM,which suggests that GSK-3β functions downstream of PTEN to antagonizemTOR activation and maintain stem cell self renewal. In support of this,GSK-3 has previously been shown to antagonize mTOR activation in HEK293Tcells by phosphorylating Tsc2 (Inoki, et al., 2006, Cell. 126:955-968).Interestingly, that work showed that Wnts could activate mTOR byinhibiting GSK-3, suggesting a bifurcation of the canonical Wnt pathwaythat could activate distinct and potentially opposing processes, asdepicted in FIG. 8. Furthermore, very recent work suggests that Wntsignaling through mTOR may also cause epidermal stem cell exhaustion,and this can also be prevented by rapamycin (Castilho, et al., 2009,Cell Stem Cell. 5:279-289). Although it is not yet known whether Wntsactivate mTOR in HSCs/HPCs, activation of both mTOR- andβ-catenin-dependent processes could explain some of the conflictingreports on Wnt effects in hematopoiesis (Staal, et al., 2008, Eur. J.Immunol. 38:1788-1794; Malhotra, et al., 2009, Cell Stem Cell. 4:27-36),if differing experimental conditions bias the effects toward eithermTOR- or β-catenin-dependent responses.

Although the Gsk3-depletion phenotype was similar to the Pten KO,important differences should also be noted. Conditional deletion of Pten(or Tsc1) leads to rapid HSC exhaustion, whereas the reduction in HSCsobserved with Gsk3-rnai became evident more slowly, through serialtransplants and competitive repopulation assays. Indeed, the initialexpansion in phenotypic HSCs was observed 4 months after primarytransplant; the number of phenotypic HSCs declined to control levels insecondary recipients, and only fell below control levels in tertiarytransplants, as depicted in FIG. 10C. It is believed that the delay isbecause the activation of HSCs and their subsequent exit from the HSCpool are balanced by enhanced Wnt signaling, which would slow the rateof HSC activation and depletion, ad depicted in FIG. 11; this idea issupported by the more rapid decline in phenotypic HSCs inGsk3-rnai,β-catenin KO BM. Pten deletion also leads to leukemia in asubstantial fraction of animals, which has not been observed so far withthe Gsk3-rnai-transplanted mice. However, PTEN regulates multipledownstream effectors in addition to GSK-3, and modulation of thesepathways could contribute to the Pten HSC phenotype independently ofGSK-3 function. It is also possible that the acute leukemia observed inPten KO mice is blocked in mice expressing nonphosphorylatable mutantsof Gsk-3.

It was found that β-catenin was required for the initial increase inphenotypic HSCs/HPCs in response to Gsk-3 inhibition and for themaintenance of Gsk3-depleted HSCs in long-term transplant assays. Theseobservations are consistent with previous studies showing thatactivation of canonical Wnt signaling can promote HSC self renewal andproliferation ex vivo (Austin, et al., 1997, Blood. 89:3624-3635; VanDen Berg, et al., 1998, Blood. 92:3189-3202; Willert, et al., 2003,Nature. 423:448-452; Reya, et al., 2003, Nature. 423:409-414; Baba, etal., 2006, J. Immunol. 177:2294-2303; Fleming, et al., 2008, Cell StemCell. 2:274-283). Although basal hematopoiesis was unaffected whenβ-catenin was deleted in adult BM, consistent with previous reports(Cobas, et al., 2004, J. Exp. Med. 199:221-229; Koch, et al., 2008,Blood. 111:160-164), inhibition of Gsk-3 can be considered a Wnt gain offunction. The issue of whether canonical Wnt signaling is required forbasal HSC homeostasis remains controversial (Staal, et al., 2008, Eur.J. Immunol. 38:1788-1794; Malhotra, et al., 2009, Cell Stem Cell.4:27-36). A recent report showed that canonical Wnt signaling canfunction in HSCs in the absence of β-catenin (Jeannet, et al., 2008,Blood. 111:142-149), which suggests that loss of β-catenin does notnecessarily block all Wnt signaling. In addition, conditional deletionof β-catenin with cre recombinase driven by the vav promoter impairsLT-HSC self renewal (Zhao, et al., 2007, Cancer Cell. 12:528-541). Asvav expression begins in utero, whereas Mx-cre was used to deleteβ-catenin in adult BM (present disclosure; Cobas, et al., 2004, J. Exp.Med. 199:221-229; Koch, et al., 2008, Blood. 111:160-164), a reasonableexplanation for these differences is that the role of Wnt/β-cateninsignaling in basal hematopoiesis depends on the developmental context.In support of an early requirement for Wnt signaling in developing HSCs,long-term reconstituting capacity in serial transplants is impaired inHSCs recovered from fetal liver of Wnt3a KO embryos (Luis, et al., 2009,Blood. 113:546-554).

Lithium's effects on hematopoiesis have been known for decades andaffect more than 90% of patients taking it, yet the mechanism of lithiumaction in this setting had not previously been defined. Plausibletargets of lithium in addition to GSK-3 include inositolmonophosphatase, which may indirectly regulate inositol trisphosphatesignaling, and related phosphomonoesterases that play important roles incell metabolism (York, et al., 2001, Adv. Enzyme Regul. 41:57-71;Gurvich et al., 2002, Pharmacol. Ther. 96:45-66). Therefore, a priori,it should not be considered obvious that GSK-3 is the biologicallyrelevant target of lithium in HSCs, and it is essential to validateGSK-3 as the target in this setting. In support of this hypothesis,structurally diverse GSK-3 inhibitors mimicked lithium effects on theHSC pool and on progenitor cells (FIG. 1; Trowbridge, et al., 2006, Nat.Med. 12:89-98; Goessling, et al., 2009, Cell. 136:1136-1147; Holmes, eta. 2008, Stem Cells. 26:1288-1297). Importantly, depletion of Gsk3a andGsk3b mimics lithium action in HSCs and HPCs as well as moredifferentiated myeloid cells. Although these observations suggest thattargeting GSK-3 may be a fruitful approach to treating hypoproliferativehematopoietic disorders, the reduction in LT-HSCs with Gsk-3 loss offunction suggests this should be approached with caution. Lithium has,in fact, been tested in clinical trials to enhance hematopoieticrecovery after myelosuppressive chemotherapy, but this approach has notseen wide use, perhaps in part because of the risk of lithium sideeffects in already critically ill patients, but also because of limitedsuccess in restoring hematopoiesis in patients with reduced numbers ofHSCs. It is also possible that the β-catenin-dependent increase in HSCsin response to lithium is offset by activation of mTOR and exit from theHSC pool, which would be consistent with the increase in moredifferentiated hematopoietic cells (especially those of myeloid lineage)commonly observed with lithium. As described herein, the combination oflithium and rapamycin in accordance with the present invention, both ofwhich are now in wide clinical use, may achieve a more marked anddurable increase in HSCs.

Example 8: Expansion of HSCs Using Gsk-3 and mTOR Inhibitors andTransplantation

It was further determined whether the expansion of HSCs exposed to atleast one GSK-3 inhibitor and at least one mTOR inhibitor increased thesuccess of HSC transplants. First, bone marrow cells were harvested fromSJL (CD45.1+) mice. The red blood cells were lysed, and c-kit positivecells were purified to enrich hematopoietic stem and progenitor cells(HSCs and HPCs). c-kit+ cells were purified according to extablishedpurification protocols and cultured in 12 well plates in (X-vivol5)medium with about 5 mM LiCl and about 5 nM rapamycin for 7 days. Nocytokines or other growth factors were added, and no serum was added.Controls included no drug added and single treatment (LiCl alone orrapamycin alone) controls. All cells from one well were divided intothree groups and transplanted to 3 lethally irradiated congenicrecipients (B6/CD45.2).

The transplant recipients that received c-kit+ cells cultured for 7 days(without added drugs) died within 14 days, similar to known controls forrecipients not transplanted with bone marrow. Transplant recipients thatreceived c-kit+ cells cultured for seven days (with LiCl) died within 18to 19 days. Significantly, 5/6 recipients of the c-kit+ cells treatedwith LiCl and rapamycin survived up to six weeks, suggesting successful,durable transplant. This indicates expansion of short-term HSCs,long-term HSCs, or both. Importantly, the transplanted cells contributegreater than 60% of peripheral blood, including myeloid and T and Bcells, at 5 to 6 weeks post transplant.

Thus, it is demonstrated herein the dual functions of GSK-3 withinhematopoietic cells. GSK-3 antagonizes the canonical Wnt pathway, andinhibition of GSK-3 activated the pathway to enhance HSC self renewal.This response to GSK-3 inhibition required β-catenin, as phenotypic HSCswere reduced in β-catenin CKO BM in both primary and secondarytransplant recipients of Gsk3-depleted BM. GSK-3 also antagonizes mTORsignaling, and inhibition of Gsk-3 either by RNAi or by conventionalgene KO activated mTOR (similar to Pten or Tsc1 KOs) and led toactivation of HSCs, with an initial expansion of LSK cells followed bydramatic depletion of HSCs (as assessed by long-term reconstitutionassays). Thus, the combination of lithium and rapamycin can be used toexpand HSCs either in vivo or ex vivo in HSC transplants, in the therapyof hypoproliferative hematologic diseases, and any other such use asdescribed herein.

Example 9: Ex-Vivo HSC Culture and Transplantation

c-kit+ cells were isolated from adult mouse bone marrow and cultured inserumfree, cytokine-free defined medium for 7 days with (triangle) orwithout (circle) our formulation. After 7 days, the entire culture wastransplanted to lethally irradiated mice (5/group) and survival wasmonitored over more than 16 weeks. All control animals (cultured withoutadditives or no transplant) died within 17 days. Mice receiving bonemarrow cells cultured in cytokine-free, serum-free medium with additivessurvived over 16 weeks. The experiment has been repeated 3 times.Erythrocytic, myelocytic, lymphocytic, and megakaryocytic lineages werepresent in peripheral blood and bone marrow after 4 months. Theseexperiments demonstrated multilineage reconstitution, and furtherdemonstrated that transplanted cells support long-term repopulation bytransplanting to a secondary recipient. The results of these experimentsare depicted in FIG. 13.

Example 10: Ex Vivo Culture of HSCs from Human Umbilical Cord Blood

CD34+ cells isolated from human umbilical cord blood have been culturedfor either 4 or 7 days in ex vivo-15 medium supplemented with GSK-3inhibitor and rapamycin, and then transplanted to non-lethallyirradiated NODSCID/NSG (immunocompromised) mice and evaluatedhuman/mouse chimerism. As depicted in FIG. 14, CD34+ cells culturedwithout cytokines but with GSK-3 inhibitor+mTOR inhibitor were aseffective in repopulating NSG mice as cells not cultured or cellscultured with cytokines. Furthermore, the cells cultured in GSK-3inhibtor+rapamycin supported long-term repopulating activity insecondary transplantation, whereas the cytokine treated cells did notsupport secondary transplants

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed:
 1. A culturing medium for expanding and maintaining ahematopoietic stem cell (HSC), comprising at least one glycogen synthasekinase-3 (GSK-3) inhibitor and at least one mammalian target ofrapamycin (mTOR) inhibitor.
 2. The culturing medium of claim 1, whereinthe culturing medium is capable of maintaining the multipotentiality ofan HSC when the HSC is maintained or expanded in the culturing medium.3. The culturing medium of claim 1, further comprising at least onecytokine.
 4. The culturing medium of claim 1, further comprising apromotion factor.
 5. The culturing medium of claim 1, wherein the GSK-3inhibitor is lithium, or a salt thereof.
 6. The culturing medium ofclaim 1, wherein the mTOR inhibitor is rapamycin.
 7. The culturingmedium of claim 1, further comprising a culture medium componentselected from the group consisting of: inorganic salts, carbohydrates,hormones, essential amino acids, vitamins, RPMI 1640, HEPES, FCS,antibiotics, and combinations thereof.
 8. The culturing medium of claim1, wherein the medium is configured to achieve an HCS expansion of about1-fold expansion to about 200-fold expansion.